Redox Remodeling by Dendritic Cells
Protects Antigen-Specific T Cells against
Oxidative Stress
This information is current as
of June 15, 2017.
Anna Martner, Johan Aurelius, Anna Rydström, Kristoffer
Hellstrand and Fredrik B. Thorén
J Immunol 2011; 187:6243-6248; Prepublished online 16
November 2011;
doi: 10.4049/jimmunol.1102138
http://www.jimmunol.org/content/187/12/6243
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Supplementary
Material
The Journal of Immunology
Redox Remodeling by Dendritic Cells Protects
Antigen-Specific T Cells against Oxidative Stress
Anna Martner,*,† Johan Aurelius,*,† Anna Rydström,*,† Kristoffer Hellstrand,*,† and
Fredrik B. Thorén*,‡
B
acterial and viral products activate the NADPH oxidase of
myeloid cells, which leads to the formation of reactive
oxygen species (ROS; or “oxygen radicals”) (1–3). ROS
are antimicrobial substances that kill ingested microbes, but they
may also compromise the function and viability of adjacent cells
when released into the extracellular space (4, 5). For example,
T cells and other lymphocytes rapidly become inactivated and
eventually undergo apoptosis when exposed to ROS (6–9). Because effector T cells commonly exert their functions in the oxidative environment characteristic of infected or inflamed tissues
(10, 11), it is conceivable that T cells use strategies to escape
oxygen radical-induced inactivation.
Earlier studies implied that molecules carrying reduced sulfhydryl groups (thiols), such as glutathione, thioredoxin, and cysteine, neutralize ROS and, thus, protect several cell types from
oxidative stress (12–14). The specific role of thiols for the function
and survival of T cells in an environment of oxidative stress is not
known. However, expression of thiols reportedly promotes T cell
function; for example, the presence of reduced intracellular thiols
(ic-thiols) in T cells and thiols expressed in cell surface proteins
*Sahlgrenska Cancer Center, University of Gothenburg, 413 90 Gothenburg, Sweden;
†
Department of Infectious Diseases, The Sahlgrenska Academy, University of Gothenburg, 413 46 Gothenburg, Sweden; and ‡Department of Hematology, The Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden
Received for publication July 26, 2011. Accepted for publication October 12, 2011.
This work was supported by the Swedish Cancer Foundation, the Torsten and Ragnar
Söderberg Foundation, the Ingabritt and Arne Lundberg Research Foundation, the
Swedish Research Council, the Gunvor and Ivan Svensson’s Foundation, the Assar
Gabrielsson Foundation, the Sahlgrenska University Hospital, and the Sahlgrenska
Academy, University of Gothenburg.
Address correspondence and reprint requests to Prof. Kristoffer Hellstrand, Sahlgrenska Cancer Center, University of Gothenburg, Medicinaregatan 1G, S-413 90 Göteborg, Sweden. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ALM-633, Alexa Fluor 633-conjugated C5maleimide; cs-thiol, cell surface thiol; DC, dendritic cell; ic-thiol, intracellular thiol;
MCB, monochlorobimane; MFI, median fluorescence intensity; ROS, reactive oxygen species; RT, room temperature; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; ViViD, LIVE/DEAD Fixable Violet Dead Cell Stain.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102138
(cs-thiols) improves T cell reactivity and proliferation (15–17).
The purported function of thiols in T cell immunity is further
bolstered by the findings that dendritic cells (DCs), which are
prominent APCs, release thiols (18, 19) and trigger thiol expression on adjacent cells, including NK cells and T cells (18, 20).
The present study sought to clarify the mechanisms and functional aspects of redox remodeling in T cells during Ag presentation, with a focus on interactions between DCs and Ag-specific
T cells. Our findings provide a novel mechanism of DC/T cell
cross-talk and point to a role for thiols in upholding the viability and
function of effector T cells in an environment of oxidative stress.
Materials and Methods
Abs and reagents
The fluorochrome-labeled anti-human mAbs used were anti-CD4 (RPAT4), anti-CD8 (RPA-T8), anti-CD69 (FN 50), anti–HLA-A2 (BB7.2),
anti-CD86 (FUN-1), anti–HLA-DR (L243) (all from BD Biosciences, San
Diego, CA); anti-CD3 (S4.1) (Invitrogen, Carlsbad, CA); and anti-TCR
Vb16 (TAMAYA1.2), anti-TCR Vb17 (E17.5F3.15.13), anti-TCR Vb22
(IMMU 546), and CMV PP65/tetramer (HLA-A*0201/PE, peptide
NLVPMVATV) (all from Beckman Coulter, Marseille, France). Purified
anti–ICAM-1 (HA58) was from BD Biosciences, and purified anti–LFA-1
(7E4) was from Beckman Coulter.
The reagents used were Ficoll-Hypaque and Lymphoprep (Nycomed,
Oslo, Norway); BSA (ICN Biomedicals, Aurora, OH); HRP (BoehringerMannheim, Mannheim, Germany); glutamate (BDH, Pool, Dorset, U.K.);
EDTA, H2O2, isoluminol, maleimide, probenecid, monochlorobimane
(MCB), 2-ME, LPS, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) (all from Sigma-Aldrich, St. Louis, MO);
Alexa Fluor 633-conjugated C5-maleimide (ALM-633), CellTracker Violet, ProLong Gold antifade stain with DAPI, and LIVE/DEAD Fixable
Violet Dead Cell stain (ViViD) (all from Invitrogen); NLVPMVATV
peptide (Biopeptide, San Diego, CA); IL-4 and GM-CSF (R&D Systems,
Abingdon, U.K.); and CryoMaxx S (PAA Laboratories, Pasching, Austria).
Cell isolation and DC generation
PBMCs were prepared from blood donor buffy coats, as described previously (3), and separated into CD14+ monocytes, CD3+ T cells, or CD19+
B cells using iMag positive selection beads (BD Biosciences), according to
the manufacturer’s instructions. The isolation procedures resulted in
.98% pure cell populations. T and B cells were immediately frozen in
CryoMaxx S. Purified monocytes were differentiated into DCs using IL-4
(500 U/ml) and GM-CSF (600 U/ml), as described (20).
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Microorganisms and microbial products induce the release of reactive oxygen species (ROS) from monocytes and other myeloid
cells, which may trigger dysfunction and apoptosis of adjacent lymphocytes. Therefore, T cell-mediated immunity is likely to comprise mechanisms of T cell protection against ROS-inflicted toxicity. The present study aimed to clarify the dynamics of reduced
sulfhydryl groups (thiols) in human T cells after presentation of viral and bacterial Ags by dendritic cells (DCs) or B cells. DCs, but
not B cells, efficiently triggered intra- and extracellular thiol expression in T cells with corresponding Ag specificity. After interaction with DCs, the Ag-specific T cells acquired the capacity to neutralize exogenous oxygen radicals and resisted ROS-induced
apoptosis. Our results imply that DCs provide Ag-specific T cells with antioxidative thiols during Ag presentation, which suggests
a novel aspect of DC/T cell cross-talk of relevance to the maintenance of specific immunity in inflamed or infected tissue. The
Journal of Immunology, 2011, 187: 6243–6248.
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DENDRITIC CELLS UPHOLD VIABILITY OF Ag-SPECIFIC T CELLS
Coculture of APCs and T cells
Results
DCs or B cells were pulsed for 2 h with SEA or SEB (0.4 mg/ml) in 96-well
round-bottom plates. SEA activates human T cells bearing Vb16 or Vb22
but not Vb17 TCR regions, whereas SEB activates Vb17+, but not Vb16+
or Vb22+, T cells (21). Ag-pulsed DCs were washed twice in the plates
with medium of ambient temperature, and autologous T cells were added
at an APC/T ratio of 1:3. In some experiments, DCs were preincubated
with 5 mg/ml anti–ICAM-1 for 40 min before T cells were added, diluting
the Ab concentration to 2.5 mg/ml in the coculture. Alternatively, T cells
were preincubated with 5 mg/ml anti–LFA-1 for 40 min before being added
to the DCs. The Ab concentration in the cocultures was 0.83 mg/ml. In
other experiments, 2 mM glutamate was added to DCs during Ag pulsing,
as well as to the DC/T cell cocultures.
For assessment of T cell proliferation, CD3+ T cells were stained with
CellTracker Violet, according to the manufacturer’s instructions, and analyzed by flow cytometry after 3–4 d of APC/T cell coculture. In some
experiments, 100 mM 2-ME was added to the cocultures.
DCs induce expression of thiols by Ag-specific T cells
Staining for thiols
CMV tetramer staining
HLA-A2+ donors with .1% of CD8+ T cells showing specific binding of
a PE-conjugated HLA-A2/NLVPMVATV tetramer (CMV tetramer) were
identified from blood donor buffy coats. DCs were pulsed with the
matching peptide (0.1 or 1 mg/ml), washed, and cultured with autologous
T cells. After 18 h, the thiol expression by Ag-specific T cells was determined by flow cytometry.
Confocal microscopy
After coculture with SEA- or SEB-pulsed DCs, 10,000 ALM-633–stained
Vb17+, Vb16+, or Vb22+ T cells were sorted out by FACS onto microscopic slides using a BD FACSAria. The purity of sorted cells was .99%.
Cells were mounted in ProLong Gold antifade stain with DAPI, and
photomicrographs were acquired using a Zeiss LSM700 confocal microscope.
H2O2 consumption assay
After overnight stimulation with APCs, activated (CD69+) and nonactivated
(CD692) T cells were sorted to .99% purity using FACS. T cells were
resuspended in Krebs-Ringer glucose buffer (500,000 cells/ml) and incubated with 100 mM H2O2 for 15 min at RT. The remaining H2O2 was
detected, as described previously (22), in a Mithras LB 940 plate reader
(Berthold Technologies, Bad Wildbad, Germany). The peak luminescence
value was normalized against the value obtained using unstimulated
T cells. In some experiments, T cells were incubated with 10 mM unconjugated maleimide in NaCl for 15 min on ice prior to the assay to
neutralize cell surface thiols.
T cell apoptosis assay
Sorted populations of CD69+ and CD692 T cells were resuspended in
complete medium (250,000 cells/ml) in the presence of H2O2. After
overnight incubation, cells were stained with ViViD, and the percentage of
apoptotic cells was determined by flow cytometry.
Statistical analysis
The expressions of thiol and CD69 by T cell populations were compared
using two-tailed paired or unpaired t tests or, for multiple populations, oneway ANOVA, followed by the Bonferroni multiple comparison test. Oxygen radical consumption and apoptosis data were analyzed by repeatedmeasures ANOVA, followed by the Bonferroni multiple comparison test
(*p , 0.05, **p , 0.01, ***p , .001).
Induction of thiols in CMV-specific CD8+ T cells after
interaction with DCs
The finding that DCs pulsed with bacterial Ags conveyed thiols to
Ag-specific T cells incited us to examine whether the thiol induction in T cells could be reproduced by DCs presenting a viral
Ag. DCs generated from HLA-A2+ blood donors with CMV
tetramer+ CD8+ T cells were pulsed with a CMV peptide
(NLVPMVATV) and cocultured with autologous T cells. The
following day, the expression of cs-thiols and the activation
marker CD69 by CD8+ T cells was determined by flow cytometry.
CMV tetramer+ CD8+ T cells were specifically activated, as determined by CD69 expression. In accordance with the results using DCs pulsed with bacterial Ags, there was a pronounced induction of cs-thiols on CMV tetramer+ CD8+ T cells but not on
nonspecific T cells (Fig. 2).
Thiols are selectively induced by Ag-pulsed DCs
We next compared the capacity of DCs and B cells to trigger Agspecific thiol expression in T cells. DCs or B cells were pulsed with
SEA or SEB and cocultured with autologous T cells. Although the
pulsed DCs and B cells induced Ag-specific CD69 expression
on T cells to a similar degree, DCs triggered significantly higher
expression of cs-thiols and ic-thiols (Fig. 3) and proliferation
(Supplemental Fig. 1C) in Ag-specific T cells. Similar results were
obtained using CD4+ and CD8+ T cells (data not shown). In accordance with these findings, the bulk of SEA- or SEB-specific
T cells (i.e., CD69+ cells) expressed significantly higher levels
of cs- and ic-thiols when activated by Ag-pulsed DCs than after
coculture with Ag-pulsed B cells (Supplemental Fig. 2).
T cells activated by DCs neutralize H2O2 and escape
oxidant-induced apoptosis
After Ag presentation in lymphoid tissue, Ag-specific T cells home
to the site of infection where they combat infection in a potentially
hostile oxidative microenvironment (10, 11). To clarify the functional consequences of DC-induced thiol expression by T cells, we
tested whether DC-stimulated T cells had acquired the capacity to
neutralize oxygen radicals. T cells that had been incubated over-
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After overnight APC/T cell incubation, T cell expression of ic-thiols, csthiols, and CD69 was assessed. Cells were incubated with 40 mM MCB
in complete medium containing 2.5 mM probenecid for 20 min at room
temperature (RT), washed, stained with ALM-633 in NaCl for 15 min on
ice, washed, and stained with surface Abs. To maintain MCB staining, all
steps were performed in the presence of 2.5 mM probenecid. Data were
acquired using FACSCanto II or FACSAria instruments (BD Biosciences),
both equipped with three laser lines (405, 488, and 633 nm), and analyzed
with FACSDiva software version 6.1.2 (BD Biosciences). Mean fluorescence intensity (MFI) values for MCB stainings were normalized against
an internal control of unstimulated T cells.
In a first set of experiments, we aimed to clarify the role of DC/
T cell interactions for the expression of thiols by Ag-specific and
nonspecific T cells. Human peripheral blood T cells were incubated
with autologous DCs presenting SEA or SEB, and the expression of
cs- and ic-thiols was determined in the respective Ag-specific T cell
subset. As shown in Fig. 1, DCs pulsed with SEA or SEB triggered
a striking expression of cs-thiols by Ag-specific T cells (i.e., cells
with TCR Vb regions corresponding to the respective bacterial
Ag). In addition to triggering cs-thiols, Ag-pulsed DCs induced icthiols in T cells with corresponding Ag specificity (Fig. 1A, 1C).
Thus, DCs pulsed with SEA triggered cs- and ic-thiol expression
by T cells with TCR Vb16 and Vb22 regions (specific to SEA),
whereas SEB trigged thiol expression by Vb17+ T cells (specific
to SEB). The Ag-specific induction of thiols was observed in
CD4+ and CD8+ T cell subsets (Fig. 1B, 1C) and was detectable
after ∼3 h of DC/T cell coculture, with sustained expression for
several days (data not shown). Fig. 1D shows confocal micrographs of cs-thiol expression by Ag-specific and nonspecific
T cells after interaction with DCs pulsed with SEA or SEB. Upon
coculture with Ag-pulsed DCs, CD4+ or CD8+ T cells with TCR/
Vb regions corresponding to the stimulatory Ag proliferated vigorously, whereas T cells with noncorresponding TCR/Vb regions did
not proliferate (Supplemental Fig. 1A, 1B).
The Journal of Immunology
6245
FIGURE 2. CMV-specific CD8+ T cells upregulate thiols in response to
Ag-pulsed DCs. CD3+ T cells were incubated overnight with CMV peptide-pulsed or nonpulsed (control) DCs. Cs-thiol expression on CMV
tetramer-positive or -negative CD8+ T cells was determined by flow
cytometry. A, Representative experiment. B, Cs-thiol expression (MFI) in
three separate experiments. **p , 0.01.
Discussion
FIGURE 1. Ag-pulsed DCs trigger Ag-specific expression of thiols by
T cells. A, CD3+ T cells were incubated overnight with SEA- or SEBpulsed DCs, and the cs- and ic-thiol expression by SEA-responsive T cells
(Vb16+ or Vb22+, green) and SEB-responsive T cells (Vb17+, red) were
determined by flow cytometry. MFI of cs-thiol (B) and ic-thiol (C) expression by Vb16+/Vb22+ and Vb17+ CD3+ T cells, CD4+ T cells, or
CD8+ T cells after overnight incubation with SEA- or SEB-pulsed DCs
(n = 13 for cs-thiols, n = 6 for ic-thiols). D, Confocal photomicrographs
of cs-thiol expression (gray) on SEA-specific (Vb16+ or Vb22+, green)
and SEB-specific (Vb17+, red) T cells after overnight culture with SEA-
Effector T cells commonly exert their functions in tissues subjected
to oxidative stress (10, 11), but the mechanisms explaining how
Ag-specific T cells survive in these hostile environments remain
incompletely understood. This study showed that DCs furnish
effector T cells with cs- and ic-thiols during presentation of viral
and bacterial Ags and that T cells with enhanced thiol expression
consume ROS and escape oxidant-induced apoptosis. In accordance with previous studies (18, 20), a slight, but significant, increase in thiol expression was observed on all T cells cocultured
with DCs. However, using Ag-pulsed DCs and detection of Agspecific TCR/Vb regions or tetramer technology, we found that
the DC-induced thiol expression was several-fold higher in T cells
specific for the presented Ag.
The conclusion that thiol induction and resistance to oxidantinduced apoptosis are more pronounced in, or even restricted to,
the Ag-specific T cell subsets is illustrated by the experiments using
SEA and SEB. DCs presenting SEA induced thiol expression
in Vb16+/Vb22+ T cells but not in Vb17+ T cells, whereas DCs
or SEB-pulsed DCs (original magnification 3126). Arrows indicate Agspecific T cells showing high thiol expression. *p , 0.05, ***p , 0.001.
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night with SEB-pulsed DCs or similarly pulsed B cells were FACS
sorted into CD69+ and CD692 populations and subsequently exposed to H2O2 as the source of ROS. As shown in Fig. 4, DCstimulated CD69+ T cells, which expressed the highest levels of
cs-thiols, were significantly more efficient consumers of H2O2
than was the corresponding CD692 subset or T cells stimulated by
Ag-pulsed B cells. Neutralization of cs-thiols, which was achieved
by the thiol-reactive reagent maleimide, efficiently reduced the
oxygen radical consumption by thiol-expressing FACS-sorted
CD69+ T cells (data not shown).
The finding that DC-stimulated T cells neutralized ROS
prompted us to investigate whether increased thiol expression by
DC-stimulated T cells conferred resistance to oxidant-induced
apoptosis. Sorted CD69+ and CD692 T cell populations were
exposed to H2O2 (100 or 200 mM) overnight, followed by assessment of T cell apoptosis. As shown in Fig. 5A and 5B, stimulation with Ag-pulsed DCs resulted in a specific protection of
T cells with Ag specificity; hence, the CD69+ T cells activated by
SEB-pulsed DCs were significantly less susceptible to oxidantinduced apoptosis than were non-SEB–specific CD692 T cells
or unstimulated T cells. CD69+ T cells activated by SEB-pulsed
B cells were not protected against oxygen radical-induced apoptosis compared with CD692 T cells or resting T cells (Fig. 5C).
6246
DENDRITIC CELLS UPHOLD VIABILITY OF Ag-SPECIFIC T CELLS
presenting SEB induced thiol expression in Vb17+ T cells but not
in Vb16+/Vb22+ T cells. Only the Ag-specific T cell subsets acquired resistance to oxidant-induced apoptosis.
Following immunization there is a pronounced increase in free
thiols in lymphoid tissue (23). These soluble thiols are assumed to
be generated, at least in part, by DCs and other APCs that release
extracellular thiols upon interaction with T cells (18, 19, 23, 24).
Uptake of cystine by DCs via the cystine/glutamate antiporter with
ensuing release of cysteine and other antioxidative thiols was
suggested to contribute to a reducing milieu in the extracellular
space (12, 18, 19). To clarify the putative role of the cystine/
glutamate antiporter for the observed induction of thiols in Agspecific T cells, we added glutamate at a concentration that efficiently reduced the cystine/glutamate exchange (19). A significant
accumulation of cs- and ic-thiols also was observed in SEA- and
SEB-specific T cells in the presence of glutamate (Supplemental
Fig. 3A, 3B), thus implying that transmembrane exchange of
cystine/cysteine is not a major mechanism accounting for thiol
FIGURE 4. T cells activated by DCs are efficient consumers of H2O2.
T cells stimulated overnight with SEB-pulsed DCs or B cells were FACS
sorted into CD69+ or CD692 populations and incubated with 100 mM
H2O2 for 15 min. Remaining H2O2 was determined using chemiluminescence. A, Representative experiment. B, Data are the mean 6 SEM of
results obtained in six donors. **p , 0.01.
induction in Ag-specific T cells. Interaction between ICAM-1/
LFA-1 was shown to be important for firm DC/T cell adhesion
(25). Treatment of DCs with Abs against ICAM-1, before and
FIGURE 5. T cells activated by DCs are protected from H2O2-induced
apoptosis. T cells stimulated overnight with SEB-pulsed DCs (A and B) or
B cells (C) were FACS sorted into CD69+ or CD692 populations and
exposed to H2O2 (100 or 200 mM) for 20 h. The percentage of T cells
undergoing apoptosis was determined by flow cytometry after staining
with ViViD. Results are the mean 6 SEM (n = 8 for DCs, n = 7 for
B cells). **p , 0.01.
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FIGURE 3. DCs are more efficient than B cells in triggering thiol expression by Ag-specific T cells. CD3+ T cells were stimulated overnight by SEA- or
SEB-pulsed DCs or B cells. A, Expression of cs-thiols and CD69 on SEA-responsive T cells (Vb16+, green) and SEB-responsive T cells (Vb17+, red). B,
Cs-thiol expression for Vb16+/Vb22+ and Vb17+ T cells (MFI; n = 13 for DCs, n = 11 for B cells). C, ic-thiol expression for Vb16+/Vb22+ and Vb17+
T cells (MFI; n = 6 for DCs, n = 3 for B cells). D, CD69 expression for Vb16+/Vb22+ and Vb17+ T cells (percentage of positive cells; n = 13 for DCs,
n = 11 for B cells). *p , 0.05, **p , 0.01, ***p , 0.001.
The Journal of Immunology
Disclosures
The authors have no financial conflicts of interest.
References
1. Betten, A., J. Bylund, T. Cristophe, F. Boulay, A. Romero, K. Hellstrand, and
C. Dahlgren. 2001. A proinflammatory peptide from Helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis. J. Clin. Invest.
108: 1221–1228.
2. Klebanoff, S. J. 1980. Oxygen metabolism and the toxic properties of phagocytes. Ann. Intern. Med. 93: 480–489.
3. Thorén, F., A. Romero, M. Lindh, C. Dahlgren, and K. Hellstrand. 2004. A
hepatitis C virus-encoded, nonstructural protein (NS3) triggers dysfunction and
apoptosis in lymphocytes: role of NADPH oxidase-derived oxygen radicals. J.
Leukoc. Biol. 76: 1180–1186.
4. Babior, B. M. 1984. Oxidants from phagocytes: agents of defense and destruction. Blood 64: 959–966.
5. Babior, B. M. 2000. Phagocytes and oxidative stress. Am. J. Med. 109: 33–44.
6. Hellstrand, K., A. Asea, C. Dahlgren, and S. Hermodsson. 1994. Histaminergic
regulation of NK cells. Role of monocyte-derived reactive oxygen metabolites. J.
Immunol. 153: 4940–4947.
7. Otsuji, M., Y. Kimura, T. Aoe, Y. Okamoto, and T. Saito. 1996. Oxidative stress
by tumor-derived macrophages suppresses the expression of CD3 zeta chain of
T-cell receptor complex and antigen-specific T-cell responses. Proc. Natl. Acad.
Sci. USA 93: 13119–13124.
8. Schmielau, J., and O. J. Finn. 2001. Activated granulocytes and granulocytederived hydrogen peroxide are the underlying mechanism of suppression of t-cell
function in advanced cancer patients. Cancer Res. 61: 4756–4760.
9. Thorén, F. B., A. I. Romero, and K. Hellstrand. 2006. Oxygen radicals induce
poly(ADP-ribose) polymerase-dependent cell death in cytotoxic lymphocytes. J.
Immunol. 176: 7301–7307.
10. Vlahos, R., J. Stambas, S. Bozinovski, B. R. Broughton, G. R. Drummond, and
S. Selemidis. 2011. Inhibition of Nox2 oxidase activity ameliorates influenza A
virus-induced lung inflammation. PLoS Pathog. 7: e1001271.
11. Nicholson, L. B., B. J. Raveney, and M. Munder. 2009. Monocyte dependent
regulation of autoimmune inflammation. Curr. Mol. Med. 9: 23–29.
12. Banjac, A., T. Perisic, H. Sato, A. Seiler, S. Bannai, N. Weiss, P. Kölle,
K. Tschoep, R. D. Issels, P. T. Daniel, et al. 2008. The cystine/cysteine cycle:
a redox cycle regulating susceptibility versus resistance to cell death. Oncogene
27: 1618–1628.
13. Ueda, S., H. Masutani, H. Nakamura, T. Tanaka, M. Ueno, and J. Yodoi. 2002.
Redox control of cell death. Antioxid. Redox Signal. 4: 405–414.
14. Kemp, M., Y. M. Go, and D. P. Jones. 2008. Nonequilibrium thermodynamics
of thiol/disulfide redox systems: a perspective on redox systems biology. Free
Radic. Biol. Med. 44: 921–937.
15. Sahaf, B., K. Heydari, L. A. Herzenberg, and L. A. Herzenberg. 2005. The
extracellular microenvironment plays a key role in regulating the redox status of
cell surface proteins in HIV-infected subjects. Arch. Biochem. Biophys. 434: 26–
32.
16. Gelderman, K. A., M. Hultqvist, J. Holmberg, P. Olofsson, and R. Holmdahl.
2006. T cell surface redox levels determine T cell reactivity and arthritis susceptibility. Proc. Natl. Acad. Sci. USA 103: 12831–12836.
17. Laragione, T., V. Bonetto, F. Casoni, T. Massignan, G. Bianchi, E. Gianazza, and
P. Ghezzi. 2003. Redox regulation of surface protein thiols: identification of
integrin alpha-4 as a molecular target by using redox proteomics. Proc. Natl.
Acad. Sci. USA 100: 14737–14741.
18. Yan, Z., S. K. Garg, J. Kipnis, and R. Banerjee. 2009. Extracellular redox
modulation by regulatory T cells. Nat. Chem. Biol. 5: 721–723.
19. Angelini, G., S. Gardella, M. Ardy, M. R. Ciriolo, G. Filomeni, G. Di Trapani,
F. Clarke, R. Sitia, and A. Rubartelli. 2002. Antigen-presenting dendritic cells
provide the reducing extracellular microenvironment required for T lymphocyte
activation. Proc. Natl. Acad. Sci. USA 99: 1491–1496.
20. Thorén, F. B., A. Betten, A. I. Romero, and K. Hellstrand. 2007. Cutting edge:
Antioxidative properties of myeloid dendritic cells: protection of T cells and NK
cells from oxygen radical-induced inactivation and apoptosis. J. Immunol. 179:
21–25.
21. Thomas, D., O. Dauwalder, V. Brun, C. Badiou, T. Ferry, J. Etienne,
F. Vandenesch, and G. Lina. 2009. Staphylococcus aureus superantigens elicit
redundant and extensive human Vbeta patterns. Infect. Immun. 77: 2043–2050.
22. Thorén, F. B., A. I. Romero, S. Hermodsson, and K. Hellstrand. 2007. The
CD162/CD56bright subset of NK cells is resistant to oxidant-induced cell death.
J. Immunol. 179: 781–785.
23. Castellani, P., G. Angelini, L. Delfino, A. Matucci, and A. Rubartelli. 2008. The
thiol redox state of lymphoid organs is modified by immunization: role of different immune cell populations. Eur. J. Immunol. 38: 2419–2425.
24. Vené, R., L. Delfino, P. Castellani, E. Balza, M. Bertolotti, R. Sitia, and
A. Rubartelli. 2010. Redox remodeling allows and controls B-cell activation and
differentiation. Antioxid. Redox Signal. 13: 1145–1155.
25. Hosseini, B. H., I. Louban, D. Djandji, G. H. Wabnitz, J. Deeg, N. Bulbuc,
Y. Samstag, M. Gunzer, J. P. Spatz, and G. J. Hämmerling. 2009. Immune
synapse formation determines interaction forces between T cells and antigenpresenting cells measured by atomic force microscopy. Proc. Natl. Acad. Sci.
USA 106: 17852–17857.
26. Corzo, C. A., M. J. Cotter, P. Cheng, F. Cheng, S. Kusmartsev, E. Sotomayor,
T. Padhya, T. V. McCaffrey, J. C. McCaffrey, and D. I. Gabrilovich. 2009.
Mechanism regulating reactive oxygen species in tumor-induced myeloidderived suppressor cells. J. Immunol. 182: 5693–5701.
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during coculture with T cells, significantly reduced the induction
of cs- and ic-thiol in Ag-specific T cells (Supplemental Fig. 3C,
3D). Hence, thiols provided during cell–cell contact in the DCT cell synapse, as well as additional signals yet to be identified,
are likely to account for the DC-induced thiol induction in Agspecific T cells.
In our study, DCs were significantly more efficacious than B cells
in triggering thiol expression in Ag-specific T cells; consequently,
only DCs conferred T cell resistance to oxidative stress. Furthermore, although DCs and B cells triggered T cell activation
(i.e., induction of cell surface CD69) to a similar extent, DCs
induced significantly more Ag-specific T cell proliferation than
did B cells (Supplemental Fig. 1C). Because thiol expression by
T cells was reported to determine their proliferative capacity (16),
it may be hypothesized that the advantage of DCs over B cells
in triggering T cell thiol expression contributes to the enhanced
T cell proliferation after stimulation by DCs compared with
B cells. The mechanism explaining the greater capacity of DCs
over B cells to induce T cell thiol expression remains to be
established, but the finding that DCs express much higher levels of
cs-thiols than B cells (Supplemental Fig. 4A) may be of relevance
for this phenomenon. We observed that the addition of soluble
thiols, in the form of 2-ME, to B/T cell cocultures only marginally
increased T cell thiol expression and proliferation (data not
shown), thus implying that the relative deficiency of B cells to
induce thiol expression and proliferation in T cells is not merely
the result of a reduced capacity to provide soluble thiols.
The expression of cs-thiols by DCs was only modestly affected
by LPS stimulation. Also, LPS activation did not significantly
enhance the capacity of DCs to induce thiol expression in Agspecific T cells (Supplemental Fig 4B). Because immature DCs
were highly efficient in inducing thiol expression in T cells, it
seems reasonable to assume that the capacity of DCs to induce
thiol expression in T cells cannot be further enhanced by maturation. These data are in accordance with a previous study
showing that immature and mature DCs confer a similar level of
protection from oxygen radical-induced inactivation to coincubated
lymphocytes (20).
The mechanisms of T cell resistance to oxidant-induced inhibition and apoptosis may be of relevance for a broad spectrum of
pathologies, including cancer, autoimmunity, and chronic infections. In cancer, ROS produced by myeloid-derived suppressor
cells are assumed to inhibit cytotoxic T cell responses (26, 27); in
contrast, a reduced ROS production in mice with an allelic polymorphism in Ncf1, which encodes a component of the ROSgenerating NADPH oxidase, entails increased T cell thiol expression, dysfunctional elimination of autoreactive T cells, and
development of autoimmunity (16, 28, 29). In several chronic infections, in particular HIV infection, there is an imbalance in the
redox status of T cells, with ensuing dysfunction and apoptosis in
T cell subsets (30–32). Collectively, these previous studies highlighted that the redox balance of T cells is critical for T cell
function and survival and point to the possibility of controlling
thiols and, hence, modulating T cell function and survival, by
pharmacological intervention (30, 32).
In summary, our results implied that DCs equip T cells with
antioxidative thiols during Ag presentation and that thiol induction
is critical for the survival of T cells in an oxidative milieu. Thiol
induction and resistance to oxidant-induced apoptosis are conveyed by DCs, but not by B cells, and restricted to the Ag-specific
T cell subset. Our findings emphasize the importance of DC/
T cell interactions for the evolvement of specific T cell-mediated
immunity and provide a novel mechanism of T cell protection
in inflamed or infected tissue.
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27. Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells as
regulators of the immune system. Nat. Rev. Immunol. 9: 162–174.
28. Olofsson, P., J. Holmberg, J. Tordsson, S. Lu, B. Akerström, and R. Holmdahl.
2003. Positional identification of Ncf1 as a gene that regulates arthritis severity
in rats. Nat. Genet. 33: 25–32.
29. Hultqvist, M., P. Olofsson, J. Holmberg, B. T. Bäckström, J. Tordsson, and
R. Holmdahl. 2004. Enhanced autoimmunity, arthritis, and encephalomyelitis in
mice with a reduced oxidative burst due to a mutation in the Ncf1 gene. Proc.
Natl. Acad. Sci. USA 101: 12646–12651.
30. Herzenberg, L. A., S. C. De Rosa, J. G. Dubs, M. Roederer, M. T. Anderson, S. W. Ela,
S. C. Deresinski, and L. A. Herzenberg. 1997. Glutathione deficiency is associated
with impaired survival in HIV disease. Proc. Natl. Acad. Sci. USA 94: 1967–1972.
31. Coaccioli, S., G. Crapa, M. Fantera, R. Del Giorno, A. Lavagna, M. L. Standoli,
R. Frongillo, R. Biondi, and A. Puxeddu. 2010. Oxidant/antioxidant status in
patients with chronic HIV infection. Clin. Ter. 161: 55–58.
32. Fraternale, A., M. F. Paoletti, A. Casabianca, L. Nencioni, E. Garaci,
A. T. Palamara, and M. Magnani. 2009. GSH and analogs in antiviral therapy.
Mol. Aspects Med. 30: 99–110.
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