The drug monosodium luminol (GVT) preserves thymic epithelial cell cytoarchitecture and allows thymocyte survival in mice infected with the T cell-tropic, cytopathic retrovirus ts1.

Abstract

A mutant of MoMuLV, called ts1, causes an AIDS-like syndrome in susceptible strains of mice. In mice infected at birth, thymic atrophy, CD4+ T cell loss, body wasting, and death occur by approximately 30-40 days postinfection (dpi). We have shown previously that the death of ts1-infected cells is not caused by viral replication per se, but by oxidative stress and apoptosis following their accumulation the ts1 viral envelope precursor protein, gPr80(env). In infected mice treated with the antioxidant monosodium alpha-luminol (GVT), T cell loss and thymic atrophy are delayed for many weeks, and body wasting and death do not occur until long after infected, untreated control mice have died. We show here that GVT treatment of ts1-infected mice maintains the thymic epithelial cell (TEC) cytoarchitecture and cytokeratin gradients required for thymocyte differentiation. It also suppresses thymocyte reactive oxygen species (ROS) levels, upregulates and stabilizes levels of the antioxidant-regulating transcription factor Nrf2, and prevents accumulation of gPr80(env) in thymocytes. We conclude that GVT treatment can make ts1 a non-cytopathic virus for thymocytes, although it cannot prevent thymocyte infection. Since oxidative stress also contributes to the loss of T cells in HIV-AIDS, the antioxidant effects of GVT may make it a useful therapeutic adjunct to HAART treatment.

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Immunology Letters 122 (2009) 159–169
Contents lists available at ScienceDirect
Immunology Letters
journal homepage: www.elsevier.com/locate/
The drug monosodium luminol (GVT®) preserves thymic epithelial cell
cytoarchitecture and allows thymocyte survival in mice infected
with the T cell-tropic, cytopathic retrovirus ts1
Virginia L. Scofield∗, Mingshan Yan, Xianghong Kuang, Soo-Jin Kim, Derek Crunk, Paul K.Y. Wong
Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Science Park - Research Division, 1808 Park Road 1-C, Smithville, TX 78957, USA
a r t i c l ei n f o
Article history:
Received 25 July 2008
Received in revised form
14 November 2008
Accepted 1 December 2008
Available online 23 February 2009
Keywords:
GVT
Thymus
Oxidative stress
a b s t r a c t
A mutant of MoMuLV, called ts1, causes an AIDS-like syndrome in susceptible strains of mice. In mice
infected at birth, thymic atrophy, CD4+ T cell loss, body wasting, and death occur by ∼30–40 days
postinfection (dpi). We have shown previously that the death of ts1-infected cells is not caused by viral
replication per se, but by oxidative stress and apoptosis following their accumulation the ts1 viral enve-
lope precursor protein, gPr80env. In infected mice treated with the antioxidant monosodium ?-luminol
(GVT®), T cell loss and thymic atrophy are delayed for many weeks, and body wasting and death do not
occur until long after infected, untreated control mice have died. We show here that GVT treatment of
ts1-infected mice maintains the thymic epithelial cell (TEC) cytoarchitecture and cytokeratin gradients
required for thymocyte differentiation. It also suppresses thymocyte reactive oxygen species (ROS) lev-
els, upregulates and stabilizes levels of the antioxidant-regulating transcription factor Nrf2, and prevents
accumulation of gPr80envin thymocytes. We conclude that GVT treatment can make ts1 a non-cytopathic
virus for thymocytes, although it cannot prevent thymocyte infection. Since oxidative stress also con-
tributes to the loss of T cells in HIV-AIDS, the antioxidant effects of GVT may make it a useful therapeutic
adjunct to HAART treatment.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
TheknownTcell-tropicretrovirusesofvertebratesincludeType
C or D retroviruses and many lentiviruses [1–4]. Some of these
agents cause leukemias and lymphomas after a months-long latent
period in their host species, while others cause fulminant dis-
eases that lead to death within weeks of infection. Interestingly,
although HIV-1 and the lentivirus simian immunodeficiency virus
(SIV) use CD4 as their surface receptor on T cells, other T cell-tropic
retroviruses do not [5–7]. Despite this, thymic atrophy, selective
infection and killing of CD4+ T-lineage cells, or neoplastic trans-
formation of thymocytes, are common characteristics of diseases
caused by these viral agents [8–14]. At present, the cause of T cell
death after infection by these viruses is unknown.
In this laboratory, we study the T cell-tropic retrovirus ts1,
whichisamutantofMoloneymurineleukemiavirus-TB(MoMuLV)
[15,16]. Because of the disease it causes, this agent falls into the
group of acute cytopathic retroviruses identified above, although
∗
Corresponding author at: Department of Carcinogenesis, The University of
Texas M.D. Anderson Cancer Center, Science Park - Research Division, PO Box 389,
Smithville, TX 78957, USA. Tel.: +1 512 237 9344; fax: +1 512 237 2444.
E-mail address: vscofield@mdanderson.org (V.L. Scofield).
its parent strain MoMuLV-TB has a long latent period, and, like
otherlong-latencyleukemiaviruses,itcausesTcellneoplasiarather
than T cell death. When used to infect newborn mice of susceptible
strains,thets1virusinducesaneurodegenerativeandimmunosup-
pressive syndrome with many features in common with HIV-AIDS
[17–21]. During its short disease course, ts1 selectively infects and
kills peripheral CD4+ T cells [19,20,22,23], although its receptor on
T cells is not CD4, but instead is the cationic amino acid receptor
MCAT-1 [5]. This CD4+ T cell loss leads rapidly to immunodefi-
ciency, wasting and death [24]. If infection is delayed for days
or weeks after birth, or if lower virus doses are used to infect
newborn pups, the latent period to disease can be prolonged
by many months, although the disease, once it develops, has a
rapid course similar to that caused earlier by higher doses of ts1
[25].
ts1 disease resembles HIV-AIDS in its latency-period spec-
trum. In the pre-HAART era of the HIV-AIDS epidemic, some
infected individuals developed full-blown disease and died within
weeks or months of primary infection, while other patients did
not develop disease for years [2]. One reason for this, for both
viruses, may involve a role for genetics in susceptibility to infec-
tion. Some inbred mouse strains are completely resistant to ts1
infection (e.g., C57Bl/6), while others are very susceptible, as is
our index strain FVB/N [15]. We suspect that the reliable and
0165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
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short timeline for ts1 disease is due to our use of FVB/N mice for
infection, our use of a standard virus dose that kills the animals
by 30–40dpi, and the young age of the animals when infected
(at birth).
In CNS astrocytes and thymocytes infected with ts1, inefficient
cleavageoftheviralenvelopepreproteingPr80envcausesittoaccu-
mulate in infected cells [26–28]. In cell types that process gPr80env
normally (yielding the two mature envelope proteins gp70 and
PrP15E), ts1 infection does not cause cell death. In cell types that do
die after ts1 infection, accumulated gPr80envinitiates an unfolded
protein response (UPR), which in turn causes endoplasmic reticu-
lum(ER)stress.TheresultisreleaseofCa2+fromcytoplasmicstores,
obligatory loading of this Ca2+into mitochondria, mitochondrial
stress, and finally by profound oxidative stress, leading to apop-
tosis [29–39]. Together our findings show that ts1-infected target
cells,whichincludeastrocytes,thymocytesandCD4+Tcells,arenot
killed by virus infection per se, but instead die by apoptosis caused
by oxidative stress [23–29].
Oxidative stress occurs in cells when the production of reac-
tive oxygen species (ROS) exceeds antioxidant defenses [40]. At
low concentrations, ROS participate in cell signaling and stimu-
late cell proliferation [41,42], but higher concentrations damage
biomolecules in the cell, leading to depletion of reduced thiols,
including the cell’s major antioxidant, glutathione (GSH) [42]. Cel-
lular defense responses to oxidative stress occur in a controlled
sequence [42]. Level 1 defenses involve upregulation of superoxide
dismutasesandcatalase,whichcounterthebuildupofH2O2formed
afterproductionofsuperoxide.IftheROSoverloadovercomesthese
defenses,thelevel2defensesaredeployedtocounteractthedeple-
tionofcysteine(aGSHprecursor)andGSHthatfollowsalargerROS
challenge. At this time, the transcription factor NF-E2 related factor
2, or Nrf2, is activated [43]. Nrf2 is the central regulatory element
controlling the transcription of level 2 cytoprotective genes, via the
antioxidant response element (ARE) sequences in their promoter
regions [43,44].
Inthisstudy,wehaverenewedourearlierworkonthelymphoid
system in the ts1-infected mouse. However, we have switched
our focus from the peripheral lymphoid system to the thymus,
and to oxidative stress mechanisms and treatments that cause or
prevent thymocyte death after ts1 infection. We chose this empha-
sis because the normal mammalian thymus is the only source of
naïve T cells, which it provides throughout life [45,46]. In HIV-
AIDS patients, naïve T cells are necessary for generation of immune
responses against new variants of HIV-1 as they arise during the
disease course. Their exhaustion has been proposed as a primary
mechanism for immunosuppression in HIV-AIDS [9,47]. As noted
above, we still have a limited understanding of how the thymus
and thymocytes are killed by cytopathic retroviruses, regardless of
the virus type or species involved [10].
This work was initiated with two objectives: first, to find
out how ts1 infection affects the thymi and thymocytes of mice,
and, second, to determine how a selected antioxidant compound
protects them. We have reported recently that the antioxidant
compound monosodium luminol (trade name GVT®) significantly
delays thymic atrophy, wasting, and death in ts1-infected mice,
even though the thymi of GVT-treated mice contain replicating
ts1 [29]. In ts1-infected mice (ts1-only), we report here that the
epithelial cell infrastructure of the thymus is destroyed, that its
thymocytes are lost to apoptosis, and that this apoptosis is accom-
panied by accumulation of gPr80env. In infected mice treated with
GVT (ts1-GVT), the epithelial cell infrastructure is maintained and
the thymocytes remain alive, although they are still infected. This
protective effect is associated with marked reduction in thymocyte
ROS levels, upregulation and stabilization of the antioxidant tran-
scription factor Nrf2 in the thymic epithelial cells (TECs), and lack
of accumulated gPr80envin the thymocytes.
2. Materials and methods
2.1. Virus
The ts1 virus, a mutant of MoMuLV, was propagated in TB cells,
a thymus-bone marrow cell line, and titered on 15F cells, as previ-
ously described [15].
2.2. Antibodies and reagents
GVT®(monosodium luminol) was provided by Bach-Pharma,
Inc., North Andover, MA. Goat anti-MoMuLV gp70 was from
Microbiology Associates, Burlingame, CA. This antibody recognizes
epitopes shared by the mature gp70 viral envelope protein and
the precursor preprotein gPr80env. Monoclonal rabbit anti-cleaved
caspase-3wasfromCellSignaling(Boston,MA).Mousemonoclonal
anti-Nrf2 was from R&D systems (Minneapolis, MN). Polyclonal
anticytokeratin-5 (CK5) and monoclonal anti-cytokeratin-8 (CK8)
antibodies were from Covance Research (Richmond, CA) and the
National Institutes of Health Developmental Studies Hybridoma
Bank (Iowa City, IA), respectively. FITC and Texas Red conjugated
anti-mouse, rat, rabbit and goat antibodies were from Jackson
ImmunoResearch (West Grove, PA).
2.3. Measurement of intracellular ROS (H2O2) using DCFDA
5 (and 6)-chloromethyl-2?,7?-dichlorodihydrofluorescein diac-
etate (CM-H2DCFDA; Molecular Probes, Eugene, OR; hereafter
called DCFDA), is a cell-permeant indicator for intracellular ROS,
including hydrogen peroxide and superoxide. The dye itself is
non-fluorescent, but when its acetate groups are removed by
intracellular esterases, it is oxidized to form a highly fluores-
cent derivative, carboxydichlorofluorescein [29]. Freshly isolated
thymocytes were prepared from uninfected, GVT-only, ts1-only
and ts1-GVT-treated mice, at 30dpi. The thymocytes were incu-
bated with 10?M of DCFDA, in culture medium (RPMI 1640)
at 37◦C for 30min. After this loading period, the cells were
washed twice with PBS, and the fluorescence of their cells
measured using a Synergy HT multidetection microplate reader
(BioTek Instruments, Inc., Winoski, VT). The data are expressed as
means±standard deviation (SD) of relative fluorescence units for
DCFDA-loaded thymocyte lysates for three mice of each treatment
group.
2.4. Mice, infection, and drug treatment
FVB/N mice were obtained from Taconic Farms (Germantown,
NY). Breeding FVB/N pairs were housed in sterilized microisolator
cages and supplied with autoclaved feed and water ad libitum. The
microisolatorswerekeptinanenvironmentallycontrolledisolation
room. For ts1 infection, 2-day-old mice were inoculated intraperi-
toneally with 0.1ml of vehicle (mock infection) or with 0.1ml of a
ts1suspensioncontaining2×107infectiousunits/ml.Miceinfected
by this protocol, and at this virus dose, become paralyzed and die
at 30–40dpi.
For GVT treatment, infected mice were divided into two groups
each on the day of infection. Infected mice were then divided again
into two groups, one of whose individuals received 0.9% normal
saline, intraperitoneally, for five continuous days a week, followed
by two resting days, until the end of the experiment, while the
other half of the infected animals received freshly prepared GVT,
delivered intraperitoneally at 200mg/kg body weight/day in 0.9%
normal saline. The uninfected mice were also divided into two
groups, one receiving saline alone, and the other receiving GVT as
described above. No toxic effects are observed at any time at this
dose of GVT when it is used without infection [29]. All mice were

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161
observed daily for clinical signs of disease, and the mice from all
groups were sacrificed at 30dpi.
Whole thymi that were to be used for Western blotting were
removed, snap-frozen in liquid nitrogen and stored at −80◦C until
use. For immunohistochemistry, thymi were snap-frozen in liq-
uid nitrogen in Optimal Cutting Temperature (OCT) embedding
compound (Sakura Finetek USA, Torrance, CA). These experimental
protocols were approved by The University of Texas M.D. Anderson
Cancer Center’s Institutional Animal Care and Use Committee.
2.5. Western blotting
Western blotting analysis was performed as described previ-
ously [29]. Briefly, proteins from whole thymic tissue or from
isolated thymocytes were extracted with radioimmunoprecipita-
tion assay buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate, 0.25mM phenylmethylsulfonylfluoride, 1mg/ml
aprotinin, leupeptin, and pepstatin A, 1mM sodium orthovana-
date, and 1mM sodium fluoride in phosphate-buffered saline, or
PBS). Protein concentrations were measured using Bio-Rad Dc pro-
tein assay reagent (Bio-Rad Laboratories, Hercules, CA) as per the
manufacturer’s instructions.
The lysates (30?g total protein per sample) were elec-
trophoresed on 10% SDS-PAGE gels, and then transferred to
polyvinylidene difluoride membranes (Millipore Corp, Bedford,
MA). The membranes were blocked at room temperature for 1h
in Tris-buffered saline, or TBS, with 5% non-fat milk, and incu-
bated with unconjugated primary antibodies for 2h, followed by 3
washes with TBS. The blots were then incubated with horseradish
peroxidase-conjugated secondary antibody for an additional 1h.
After three washes, immune complexes were detected by chemilu-
minescence(NENLifeScienceProducts,Boston,MA).Amonoclonal
anti-?-actin antibody (Sigma, St. Louis, MO) was used as a loading
control. In Western blots for which fold differences of bands were
calculated, the densitometry readings for the bands were first nor-
malized against the ?-actin densities for the same lanes, and then
the ratio obtained from this calculation set as 1 for bands for cells
or tissues from uninfected mice, and the other values calculated as
the ratio of their normalized values against this number.
2.6. Immunohistochemistry
Dissected tissues were snap-frozen in OCT medium and kept at
−80◦C prior to cutting of 5-?m-thick serial frozen sections. The
sections were placed on microscope slides, and the slides kept at
−80◦C prior to staining. For staining, the sections were thawed at
room temperature for 30min, fixed in ice-cold acetone for 5min,
washed, and incubated overnight at 4◦C with optimum dilutions
of primary antibodies. The slides were then washed and incubated
in FITC-conjugated or TxR-conjugated anti-rabbit, anti-rat, or anti-
goat IgG Fab?2.
Where double immunostaining was done, it was with poly-
clonal or monoclonal antibodies raised in cells or animals from
two different species, followed by FITC-conjugated anti-IgG for one
species, and TxR-conjugated anti-IgG for the other. After incuba-
tion in the secondary reagents and washing, the sections were
mounted in mounting medium (Vector Laboratories, Burlingame,
CA) for viewing under an Olympus fluorescence microscope. Con-
trol sections were incubated (a) in affinity-purified goat, rabbit or
rat IgG, depending upon the host species of the primary antibody,
(b) with appropriate isotype controls, for monoclonal antibodies,
(c) in secondary antibodies alone, without primary reagents. Opti-
mum staining conditions were developed for each antibody, so that
no specific binding was observed in sections incubated in control
reagents, while specific binding was observed with the primary
antibodies under test.
2.7. Statistical analysis
Western blotting experiments were repeated at least three
times, and immunostaining studies were repeated at least twice, to
verify reproducibility of results. Quantitative differences between
groups in graph-displayed data were compared for statistical sig-
nificance by Student’s t-test, and p-values of less than 0.05 were
considered statistically significant.
3. Results
3.1. Epithelial cell organization is disrupted in the thymi of
ts1-infected mice, but maintained in infected mice treated with
GVT
In the normal mouse thymus, the epithelial matrix of the organ
is divided into grape-like lobules, each of which is composed of a
well-demarcated outer cortical epithelium and an inner medullary
epithelial cell area. Both areas are like epithelial cell “baskets” filled
withthymocytesandothercelltypes,includingmacrophages,den-
dritic cells, fibroblasts, and a rich connective tissue matrix. In each
lobule, T cell precursors arrive in the thymus from the bone mar-
row, landing in the corticomedullary junction (CMJ) between the
cortexandmedulla[48].ThesubsequentdifferentiationoftheTcell
precursorsintomatureTcellsthenoccursinthecontextofthiscor-
ticomedullary structure, during which time the thymocytes travel
fromtheCMJthroughthecortextotheouteredgeofthelobule,and
then come back again, all the while undergoing a scheduled differ-
entiationsequencethattakesthemthroughdefineddevelopmental
checkpoints. These events are governed by contact-mediated and
humoral reciprocal crosstalk between the TECs and thymocytes
[49,50].
Theepithelialthymuscontainstwodistinctkindsofcells:asim-
ple epithelium in the cortex, and a basal epithelium in the medulla.
Normal corticomedullary organization of the murine thymus has
a molecular component that can be assessed using antibodies to
cytokeratinmarkers.Cytokeratinsareadiversefamilyofintermedi-
ate filament proteins that distinguish different epithelial cell types,
including the two epithelial cell types in the mouse thymus. In the
healthy mouse, the cortical epithelial cells express cytokeratin-8
(CK8), the medullary epithelial cells express cytokeratin-5 (CK5),
and a small population separating in the CMJ expresses both
[49]. The presence of an intact epithelial cell cytokeratin CK8/5
expression gradient, and of a clear corticomedullary boundary in
H&E-stained slides, is a precise anatomical correlate of normal thy-
mocyte differentiation [49,50].
In the mouse thymus, TEC differentiation is dysregulated if the
thymocytescannotdifferentiate;likewise,normalthymocytescan-
not differentiate in thymi whose epithelium is abnormal (reviewed
in [49]). To determine how ts1 infection affects the epithelial
and thymocyte compartments in the thymus, and to follow GVT
effects on these events, we first compared cytoarchitecture and
cytokeratin expression patterns for TECs and thymocytes in unin-
fected, ts1-infected (ts1-only) and infected GVT-treated (ts1-GVT)
mice sacrificed at 30dpi. Frozen sections from thymi of uninfected
and ts1-infected (ts1-only) and ts1-infected, GVT-treated (ts1-GVT)
mice were prepared at 30dpi, and then (1) stained with H&E, (2)
doubly immunostained either with anti-CK8 and anti-CK5 (on an
uninfected thymus section), or (3) doubly stained with anti-gp70
and CK8 (infected ts1-only or ts1-GVT thymic sections). The left
side panel in Fig. 1(A) is an H&E-stained section of thymus from an
uninfected mouse. It shows clear lobular cortimedullary epithelial
organization,andtheleftimmunostainedsectioninFig.1(B),which
also is from an uninfected mouse thymus, shows correct epithe-
lial cytokeratin organization (CK8-positive cortex, CK5-positive

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Fig. 1. Corticomedullary epithelial organization and distribution of epithelial cell cytokeratins are dysregulated in 30dpi ts1-only mice, but these disturbances are prevented
ints1-GVTmice.Thewhiteboxesinthets1-onlyandts1-GVTH&E-stainedsectionsin(A)correspondtotheimmunostainedsectionsbelowthemin(B).Theuninfectedthymus
section at the left in (A) shows three lobules with normal corticomedullary organization, while the lobule in the ts1-only thymus section has disorganized corticomedullary
organization, with a medullary epithelium whose cells have spread into the cortical subcapsular space. By contrast, the section from the ts1-GVT mouse thymus shows
normal corticomedullary organization. The uninfected immunostained thymic lobule at the left in (B) has normal corticomedullary cytokeratin organization, in which
cortical epithelial cells are CK8-positive (green), medullary epithelial cells are CK5-positive (red/orange) and the epithelial cells of the CMJ are positive for both (yellow cells;
arrow). The ts1-only, gp70/CK5-immunostained section at the center in (B) contains infected, gp70-positive thymocytes (green) and infected, gp70-positive and CK5-positive
epithelial cells (yellow). As in the ts1-only H&E section above in (A), the medullary epithelial cells appear to be expanding out of the medullary space, and no distinct CMJ is
evident. From their uniform staining with both antibodies, all of these epithelial cells appear to be infected. The ts1-GVT gp70/CK8-immunostained section at the right in (B)
contains infected, gp70-positive thymocytes and CK8-positive cortical epithelial cells. Some of the CK8-positive cells are also gp70-positive, by their yellow co-staining), but
most appear to be singly CK8-positive, and thus may not be infected. This section also has a clearly demarcated CMJ (separating the lower right of panel from the upper left).
Original magnifications: (A) all panels 4×; (B) left panel, 4×; middle panel, 10×; right panel, 20×.
medulla). In the ts1-only sections in the middle panel of Fig. 1(A),
severe cortical thinning is evident in the H&E-stained section, and
the corresponding immunostained section in Fig. 1(B) (see box in
H&Esection)showsthatCK5-positivemedullaryepithelialcellsare
not confined to the medulla as they are in the normal thymus, but
instead are growing out into the thinning thymic cortex. Fig. 1(A)
and (B) also show sections from the thymic cortex of a 30dpi
ts1-GVT mouse (right). The H&E stained section has normal cor-
ticomedullary organization. The immunostained ts1-GVT section
(alsoseeboxinFig.1(A))containslarge,apparentlyuninfected(red,
not yellow) CK8+ TECs within in an intact CMJ boundary (extend-
ing from lower left to upper right in the panel). In the same section,
gp70-positive thymocytes surround the TECs. The next question,
therefore, was: are the TECs and thymocytes dead in the thymi of
ts1-only mice, and alive in the thymi of ts1-GVT mice?
3.2. Apoptosis is elevated in TECs and thymocytes of thymi
ts1-infected mice, but not in infected mice treated with GVT
Caspase-3istheexecutiveenzymethatcompletesapoptoticcas-
cades in most cell types. The presence of the activated (cleaved)
formofcaspase-3isaspecificindicatorofapoptoticcelldeathintis-
sues,includingthethymus[51].Inthets1-onlyandts1-GVTsections
stained for CK8 and gp70 in Fig. 1(A) and (B), ts1-infected thymo-
cytes and TECs are visible, but the images cannot tell us whether
these cells are alive or dead. To find out, we compared levels of
activated caspase-3 protein in thymocytes from these animals. The
Western blot in Fig. 2(A) shows that ts1-only thymocytes contain
elevated amounts of cleaved caspase-3, relative to uninfected thy-
mocytes, while ts1-GVT thymocytes have somewhat less cleaved
caspase-3,inrelationtothets1-onlytissues.Itshouldbenotedthat
the relatively high levels of cleaved caspase-3 in uninfected thy-
mocytes are normal, because most T cells generated in the thymus
also undergo apoptosis there [45,46]. Thus, if the ts1-GVT thymus
is functioning normally, cleaved caspase-3 levels should approach
those of uninfected thymi, as they apparently do (Fig. 2(A)).
For an in situ look at caspase-3 in the thymi the three groups
above, we stained 30dpi thymic sections for gp70 or for cleaved
caspase-3. The photomicrographs in Fig. 2(B) show that cells posi-
tive for gp70 are abundant in thymi from bothts1-only and ts1-GVT
mice.However,whilecleavedcaspase-3-positivecellsareabundant
in the ts1-only thymus section, they are apparently absent in the
ts1-GVT section (Fig. 3(C)).
3.3. Thymocyte numbers and weight are dramatically reduced in
the ts1-only thymus, but not in thymi of infected mice treated
with GVT
The loss or presence of cytokeratin-based corticomedullary
organization in ts1-only thymi (Fig. 1), and the presence or absence
ofcleavedcaspase-3inthesetissues(Fig.2)arereliablebutindirect
markers of dysregulated (ts1-only) vs. apparently normal (ts1-GVT)
T cell differentiation. To assess the state of T cell differentiation
directly,wecomparedthetotalnumbersofthymocytes,andthymic
weights, for 30dpi mice from the uninfected, GVT-only, ts1-only,
and ts1-GVT treatment groups. The bar graphs in Fig. 3(A) show
that statistically significant thymocyte loss is evident in 30dpi
mice from the ts1-only group, but not in the thymi of ts1-GVT
mice (p<0.001). Similarly, when we compared average thymus
weights for the same mice, prior to isolation of their thymocytes,

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Fig. 2. Cells of the thymus die by apoptosis in ts1-only mouse thymi, but not if the thymi are from ts1-GVT animals. (A) Western blot of thymocyte lysates for comparison of
their contents of activated caspase-3. The ts1-only thymocytes contain more of this enzyme than do uninfected thymocytes, while ts1-GVT thymocytes contain intermediate
amounts. ?-actin was used as a loading control. (B and C) Sections of thymi from mice of the three groups in (A), stained either for gp70 (green; to detect ts1 infection) and
for cleaved caspase-3 (red). Both the ts1-only and the ts1-GVT sections have gp70-positive infected cells, but only the ts1-only section has cells that are positive for cleaved
caspase-3. The sections from the ts1-GVT thymus are both from the thymic cortex; this may explain the unexpected absence of caspase-3-positive cells in the right lower
panel in (C), given the presence of at least some cleaved caspase-3 in the thymic tissues of ts1-GVT mice. Original magnifications (A and B): all panels, 20×.
we observed, as expected, a significant loss of thymus weight in the
30 thymi of ts1-only mice (Fig. 3(B); p<0.001). However, no thymic
weight loss had occurred in 30dpi infected mice if they had also
been treated with GVT. A small but significant weight increase was
evident in the ts1-only thymi, relative to the uninfected controls
(p<0.05).
3.4. ROS levels are reduced in thymi from GVT-treated mice
We next asked how GVT prevents thymocyte apoptosis in ts1-
GVT mice, whose TECs and thymocytes are infected but alive at
30dpi (Figs. 1–3). We had reported previously that oxidative stress
isthetriggerforapoptosisofts1-infectedastrocytes,andthatoxida-
tive stress also occurs in thymi of ts1-infected mice, but that GVT
treatmentpreventsbothastrocyteandthymocytedeathinsitu[29].
We therefore asked whether GVT treatment of mice actually affects
the intracellular redox conditions in their thymocytes, using the
fluorescent dye DCFDA to quantitate their contents of ROS [52].
The bar graphs in Fig. 4 show that thymocytes from GVT-treated
mice do have significantly lower ROS contents than thymocytes
from uninfected mice, whether the mice are infected or uninfected
or not (p<0.001, when compared either to uninfected or ts1-only
thymocytes). These observations tell us two new and significant
things: one, the ROS-lowering effects of GVT do not require con-
comitant infection with ts1 or other oxidant stressors, and, two,
that GVT treatment somehow sets up conditions in which a steady-
state redox “tone” is established and maintained in thymocytes of
infected treated animals, thus presumably affecting all intracellu-
lar parameters impacted by ROS levels in the cells. These results
link GVT protection of the thymus to events that reduce ROS levels
in thymocytes, and they suggest that GVT protects thymocytes by
setting up low-ROS conditions that allow ts1 infection of thymo-
cytes, but prevent the cytopathology and apoptosis that otherwise
would kill the infected cells. How might these low-ROS conditions
be established in the thymocytes by GVT?
3.5. Nrf2 upregulation and stabilization in TECs of thymi from
GVT-treated, infected mice
To determine how GVT might cause lowering of redox setpoints
in thymocytes, we took into account results from our earlier pub-
lishedstudiesusingculturedastrocyticcells[29,37,38].Whenthese
cells were infected in culture with ts1, about 50% of the cells died,
but 50% remained alive, and these could be passaged over a long
period of time [38]. Notably, the astrocytes that survived were dif-
ferent from those that died, with respect to (a) their maintenance
of low steady-state ROS levels, and (b) their high levels of Nrf2 [38].
These results suggested that the high Nrf2 levels in ts1-infected
astrocytes were causally linked to their low ROS.
Many cell types, including astrocytes, maintain ambient levels
of the Nrf2 protein, which is a transcription factor that coordinately
upregulates many genes that participate in Phase 2 antioxidant
defenses. In non-stressed cells, Nrf2 is held in an inactive state as
partofacomplexwiththeKeap-1molecule.Aslongassteady-state
conditions prevail, the complexed Nrf2 is cyclically ubiquitinated,
releasedtotheproteasomesfordegradation,andreplacedbynewly

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Fig. 3. Total numbers of thymocytes and average thymic weights in 30dpi thymi
from uninfected, GVT-only, ts1-only, and ts1-GVT mice. (A) The bar graphs show
the means±standard deviations for total numbers of thymocytes for five mice from
each group.**p<0.001 for ts1-only mice vs. each of the three other groups. (B) The
bar graphs show the average weights for thymi of the four groups in (A).**p<0.001
for ts1-only mice vs. each of the three other groups;*p<0.05 when compared to
uninfected thymi.
Fig. 4. Thymocyte ROS contents are reduced in mice treated with GVT, whether
or not they are infected. The bar graphs show the average of relative numbers of
fluorescenceunitsofDCFDAinlysatesofthymocytesfromuninfected,GVT-only,ts1-
only and ts1-GVT mice, represented as the mean±standard deviations for values for
three mice of each group.**p<0.001 for thymocytes of GVT-treated mice (infected
or not) vs. thymocytes from uninfected or ts1-only mice.
translated Nrf2 [53–55]. When oxidant stress conditions occur,
however, redox-sensitive sites on the Keap-1 protein allow it to
release Nrf2, which is then phosphorylated and transported to
the nucleus. In the nucleus, Nrf2 activates genes that have ARE
sequences in their promoters (reviewed in [54]).
When ROS levels in the cells are returned to normal, it has been
assumed that the activated Nrf2 that is left is ubiquitinated and
degraded. However, our results for astrocytes [38], and recent work
by other laboratories, have shown that Nrf2 levels can be stabi-
lized in the cytoplasm and nucleus under certain kinds of oxidant
stress conditions [56], or by drugs that inhibit Nrf2 degradation in
proteasomes [57,58]. Agents that stabilize Nrf2 levels in cells have
beenidentifiedashavinganti-inflammatoryeffects,mostlikelydue
to the continued or amplified antioxidant defenses that might be
availabletocellsasaconsequence[57].Wethereforeaskedwhether
Nrf2upregulationandstabilizationoccursinthymocytesofts1-GVT
mice.
The results of these experiments are in Fig. 5. Surprisingly and
againstourexpectations,ourfirstWesternblotsforNrf2,performed
with isolated thymocytes, show that Nrf2 levels of ts1-only and
ts1-GVT mice are only slightly upregulated over levels in unin-
fected thymocytes (Fig. 5(A)). This result was also obtained in an
earlier publication from this laboratory [29]. To find out whether
Nrf2 upregulation instead occurs in other cells in the thymus, we
performed a second Nrf2 Western blot, this time using whole thy-
mus tissue. The results in this Western blot in Fig. 5(B) indicate that
whole thymus tissues from ts1-only and ts1-GVT mice have a two-
fold increase in amounts of Nrf2, over those of uninfected cells.
We conclude that thymocytes of tsl-only or ts1-GVT mice do not
change their Nrf2 levels, but that other cells in the thymus do. This
result poses a different scenario from the one we had constructed
when designing this experiment, in which thymocytes of ts1-GVT
mice protect themselves by upregulating their Nrf2 levels, as do
astrocytes that survive ts1 infection [38]. If Nrf2 plays a role in low-
ering ts1-GVT thymocyte ROS levels, and if low-ROS conditions are
responsible for the survival of these cells, then Nrf2 upregulation
and stabilization may occur in cells that support thymocyte redox
homeostasis, rather than in thymocytes themselves.
There are many non-thymocyte cell types in the normal thy-
mus, including dendritic cells, macrophages, and TECs [59]. Others
have shown that dendritic cells control T cell activation events via
the T cell receptor, by regulating the redox environment of antigen
presentation and recognition [58]. In thymi of ts1-GVT mice, the
cells most likely to play a supporting role of this type would be the
TECs. In addition to hosting thymocyte differentiation, TECs may
provide metabolic and redox support to thymocytes via Nrf2 stabi-
lization and release of thiol redox antioxidants to thymocytes, just
as astrocytes do for neurons in the CNS under oxidant stress [60].
Our early published work has shown that thymocytes of FVB/N
mice can be cultured and infected by ts1 in vitro, but only if IL-
2 and IL-7 (produced by thymocytes and TECs, respectively) are
added to the culturing medium, and only if the culturing wells con-
tain thymic remnants [61]. More recent studies with HIV-1 have
produced similar results, in showing that TECs must be present for
infection of cultured thymocytes [62]. For ts1 and HIV-1 infection
of cultured thymocytes, therefore, TECs may be required to make
virus available for thymocyte infection, or to provide metabolic or
redox support for infected thymocytes. For the same reasons, GVT
protection of thymocytes may require the presence of TECs that
can upregulate and stabilize their Nrf2. To determine whether this
might be the case, we first prepared frozen sections of uninfected
ts1-only vs. ts1-GVT thymi, and doubly stained them with gp70
and Nrf2. The photomicrographs in Fig. 5(C) show that the ts1-only
thymic section contains many large, infected gp70-positive cells,
all of which also contain Nrf2 (no red cells are present, indicat-
ing that all Nrf2-positive cells are also infected). By contrast, the

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165
Fig. 5. Levels of the transcription factor Nrf2 are not significantly elevated in ts1-only or ts1-GVT mouse thymocytes at 30dpi, but are in whole thymic tissues, where the
proteinisconcentratedinnon-thymocyticcells.(A)WesternblotofthymocytelysatescomparedfortheiramountsofNrf2.Folddifferencecalculationsdisclosednosignificant
differences were evident among uninfected, ts1-only and ts1-GVT thymocytes. (B) Western blot of whole thymic tissue lysates from animals of the same three treatment
groups. Amounts of Nrf2 in the ts1-only and ts1-GVT tissues were at least twice those in uninfected tissues, indicating that some cell type(s) in the thymus do upregulate their
Nrf2 levels after ts1 infection in ts1-only and ts1-GVT thymi. (C) Sections of thymi from mice of the three different treatment groups in (A) and (B), doubly immunostained
for gp70 (green, for infection) and for Nrf2 (red). In the ts1-only section, not all gp70-positive infected cells are yellow (co-stained for both markers), but all Nrf2-positive
cells are. By contrast, sections of thymi from ts1-GVT mice contain both gp70-positive (infected cells) and Nrf2-positive cells, but the cells stained by the two antibodies are
largely either green or red, but not yellow, indicating that they express either gp70 or Nrf2, but not both.
thymic section from the ts1-GVTmouse shows many gp70-positive
infected cells (green; without Nrf2), but also has large numbers of
red cellular profiles (Nrf2-positive only) that are not thymocytes.
To determine whether these Nrf2-positive cells are TECs, we
stained one ts1-only and one ts1-GVT section for Nrf2 and CK8.
Fig. 6(A) shows that all of the Nrf2-positive cells in ts1-only sec-
tion are CK8-positive cortical epithelial cells, but that these cells
lack organization and appear to be dying or dead. In the ts1-GVT
section, however (Fig. 6(B)), the epithelial cells are intact, and all
of the cells are positive for Nrf2 and CK8 (yellow). We conclude
that TECs upregulate and stabilize their Nrf2 in ts1-GVT thymi, and
that this TEC loading with Nrf2 may maintain low-ROS conditions
in their thymocytes, most likely by providing the thymocytes with
cysteine substrates or GSH.
3.6. gPr80envis processed normally in thymocytes of infected,
GVT-treated mice
One possible consequence of low-ROS conditions in thymocytes
of ts1-GVT mice might be the normal processing of gPr80envto
gp70 and PrP15E in infected thymocytes. As noted above, we have
established that ts1-infected astrocytes and thymocytes die as a
consequenceofaccumulationofthemutantts1preproteingPr80env
in the ER. Accumulation of gPr80envtriggers the UPR, which in
turn elicits ER stress and calcium loading into mitochondria, caus-
ing mitochondrial and oxidant stress-induced apoptosis [29–38].
In normal cells, the local redox environment in the ER is controlled
to provide reducing conditions necessary for formation of disul-
fide bonds, and to promote or inhibit hydrogen bonding during
the translation of proteins with complex folding requirements[63].
We therefore wondered whether the low-ROS conditions in ts1-
GVT thymocytes might allow normal folding and processing of the
ts1 mutant gPr80env. This would protect them from the apoptotic
cascades that are activated in thymocytes of ts1-only mice.
To determine whether this occurs, we used Western blotting
with anti-gp70 to compare amounts of gPr80env(uncleaved) to
gp70 (cleaved) envelope protein in thymocytes of ts1-only vs. ts1-
GVT mice. The results in Fig. 7 show that ts1-only infected tissues
contain both aggregated gPr80envat 80kDa, and processed gp70, at
70kDa. This is a typical Western blotting result from ts1-infected
astrocytes and thymocytes, whose processing of gPr80envis ineffi-
cient [26–29]. By contrast, all of the gp70-immunoreactive protein
is in the 70kDa band from thymocytes of ts1-GVT mice, indicating
that the gPr80envis being processed normally in these cells.
4. Discussion
Like ts1, other cytopathic retroviruses, including HIV-1, SIV and
FIV, cause damage to thymic cytoarchitecture and loss of thymo-
cytes in their respective host species, making the thymus unable
to supply naïve T cells for protective immune responses to viral
variants as they appear during the disease course [8–10,13,63–65].
HAART therapies, like those currently in use for treatment of HIV-
AIDS, may not restore thymopoiesis even assuming that normal
Tprogenitor cells are produced and sent to the thymus from the
bone marrow in these patients [66]. This means that naïve T cell

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V.L. Scofield et al. / Immunology Letters 122 (2009) 159–169
Fig. 6. The Nrf2-positive cells in ts1-only and ts1-GVT thymi are CK8-positive TECs. The thymic section in (A) was doubly stained with anti-gp70 (green) to detect infection,
and with anti-Nrf2 (red). The merged panel from these images, at the right, shows a generally disorganized tissue containing TECs and thymocytes, without a clear pattern of
co-staining for the two. By contrast, the thymic section in (B), which was stained in the same way, contains a clear Nrf2-defined area, with CK8-positive cells on the left and
right side (line in all panels), and Nrf2-positive cells on the left side. The arrows in all panels in (B) point to a CK8-positive, Nrf2-positive cortical TEC.
exhaustion, thought to be one cause of HIV-1 immunosuppres-
sion [9–11] might still occur under HAART treatment. For the same
reasons, bone marrow or hematopoietic stem cell transplantation,
once considered as potential treatments for T cell exhaustion in
HIV-AIDS [66–68], may not repopulate the thymus either, unless
there is a functioning and non-toxic (e.g., low-ROS) microenviron-
ment there in which the stem cells can differentiate to become T
cells.
The results of this study may address some of these problems,
although they come from studies of what happens to the mouse
thymus infected by only one type of T cell-tropic retrovirus. The
work reported here confirms that TEC and thymocyte loss follow-
ing ts1 retroviral infection are not the results of infection per se,
but rather are the result of oxidative stress leading to apoptosis of
thymocytes. If oxidative stress also occurs in the HIV-AIDS thymus
and thymocytes, and if GVT can reduce or eliminate it even as the
cells remain infected, then repopulation of the thymus might be
possible in these patients, either by autologous bone marrow T cell
progenitors or by transplanted bone marrow stem cells [66–68].
GVT is an antioxidant compound that has anti-inflammatory
and anti-apoptotic properties in many animal and human diseases
Fig. 7. Western blot of 30 dpi thymic tissues from uninfected, ts1-only and ts1-
GVT mice, probed for gp70. The antibody identifies epitopes present in both the
band present at 80kDa, which is gPr80env(the viral preprotein), and in the band
at the 70kDa position (gp70). This blot shows that gPr80envaccumulation occurred
in this ts1-only thymus, but not in the thymic tissues of the ts1-GVT mouse whose
tissueswereusedforthisblot.Somets1-GVTthymicontaingPr80envat30dpi,butin
significantly smaller amounts than those seen for thymi of ts1-only mice, and with a
correspondingly larger gp70 band, than those seen for ts1-only thymi (not shown).
[29,69–73]. GVT is also a non-toxic drug in mice [29] and humans
[69–73]. When we started these studies, we already knew that GVT
protects the thymi of ts1-infected mice, both at the gross level (by
maintaining normal thymus weight) and the cellular level (by pre-
venting oxidative stress in thymic cells) [29]. In ts1 infection, the
thymus appears to be a “staging area” from which infectious virus
is disseminated to other tissues, including the CNS; in fact, a thy-
musmustbepresentifCNSandthensystemicinfectionaretooccur
[74]. The simplest way to interpret the systemic protective effect
of GVT in ts1 infection, then, would start with the thymus. The
survival and apparent homeostasis of its TEC and thymocyte cell
compartments, under GVT treatment, could be the basis of what
becomes either a fatal disease or a harmless infection in other tis-
sues as well (including the intestine [97]). In the ts1-GVT thymus,
we have shown here (1) that the TECs maintain normal character-
istics in ts1-GVT mice; (2) that thymocytes of ts1-GVT mice survive
despite continued infection; (3) that the thymocytes have lower
than normal ROS levels; (4) that TEC Nrf2 levels are upregulated
and stabilized; and (5) that these thymocytes do not contain accu-
mulated gPr80env. If the first of these is causally linked in series to
the last, then the prosurvival effects of low ROS conditions, caused
by GVT, could either be direct (via GVT alone) or indirect, via GVT
upregulation and stabilization of Nrf2. A diagram showing these
possible pathways is provided in Fig. 8.
Given that other retrovirus infections involve oxidative stress
(including HIV-1 and SIV; see below), it is tempting to speculate
thatGVTtreatmentcouldpreventthymocytelossandmaintainnor-
mal thymic cytoarchitecture in all cytopathic retroviral diseases. As
noted above, we believe that the ts1-infected mouse is a model
for HIV-AIDS, despite differences between the two viruses and the
diseases they cause [16–23]. Matching characteristics between the
ts1-infected mouse and the HIV-infected human have been iden-
tified in published papers from our laboratory and from others
[16,19–21]. The ts1 virus selectively infects and kills CD4+ periph-
eral T cells [18,19,22,23] and CD4+ thymocytes (V.L. Scofield et al.,
in preparation).

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Fig. 8. Diagram showing possible mechanisms for protection of 30dpi ts1-infected
mouse thymi and their thymocytes by GVT. The arrows point to processes initiated
or promoted by ts1 infection or GVT treatment, and the T-bars identify GVT inhibi-
tion or termination of processes initiated by ts1 infection in T cells. In the ts1-only
mouse, both TECs and thymocytes are damaged. The result is the disappearance
of epithelial cell gradients, loss of thymocytes, and thymic atrophy. In the ts1-GVT
mouse, the epithelial cells and thymocyte compartments appear normal, although
the TECs accumulate and stabilize their Nrf2, and the thymocytes have lower-than-
normal ROS levels. The thymocytes and TECs survive infection, and gPr80envdoes
not accumulate in infected thymocytes. As for the intestine [97], our data suggest
that this protection could occur either in two ways: via direct antioxidant activity
by GVT, or lowering of thymocyte intracellular redox setpoints by gluthione (GSH)
or by GSH precursors, both produced and provided to the thymocytes by TECs as a
consequence of their upregulation of Nrf2.
Although it might be argued that the abnormal protein accumu-
lation and oxidative stress that kill ts1-infected thymocytes do not
participate in T cell loss in HIV-1 infection, we would respond by
pointing out that this question has not been experimentally tested
in a direct way. Given that the mechanisms leading to thymocyte
and T cell death in HIV-AIDS are still not known [2,10], we would
suggest that this issue now be the object of concentrated investi-
gation in HIV-AIDS. Dozens of studies over the past decades now
suggest that abnormal viral protein accumulation [75–78] and/or
oxidative stress [79–93] occur in loss of T cells in HIV-AIDS. Recent
work has shown that folding of nascent membrane proteins in the
ER of healthy cells depends upon a carefully controlled intracellu-
lar redox environment [63,78,94]. In cells whose ROS levels have
risen due to oxidant challenge, misfolding theoretically is a pos-
sibility for all membrane proteins in the cell. In light of this, it is
interesting to note that the HIV-1 membrane preprotein gp160 has
particularly strict requirements for sequential and complex fold-
ing events [78], and that these make it especially likely to misfold
and aggregate when ROS levels increase in infected cells. Since ts1
gPr80envmisfolds in thymocytes whose ROS levels are near nor-
mal or only slightly elevated (Figs. 4 and 7), the lowering of ROS
caused by GVT treatment in thymocytes of ts1-GVT mice may pro-
mote normal folding of gpr80env. If this occurs, it may be why TECs
and thymocytes in ts1-GVT mice survive infection (Fig. 8). We hope
that the findings reported here will re-awaken the possibility that
abnormalproteinaccumulation,followedbyoxidativestress,might
leadtothekillingofthymocytesandTcellsinHIV-AIDSandinother
retroviralsyndromesthattargetthesecells.Ifso,thenGVTandother
antioxidant compounds may allow maintenance or restoration of
a normal thymic microenvironment in HIV-AIDS patients, allowing
thedifferentiationofautologousbonemarrowTcellprogenitors,or
of T cell progenitors from bone marrow or stem cell allotransplants
[95,96].Asanantioxidant,GVTmayprovetohavewidertherapeutic
utility as a non-toxic anti-inflammatory drug that exploits natural
antioxidant mechanisms (e.g., Nrf2) to prevent tissue damage after
oxidant injury.
Acknowledgements
This paper is dedicated by the first author to the memory of
Professor John J. Marchalonis (1941–2007). We thank the late Dr.
David Klug for important discussions, and we acknowledge indis-
pensable help and advice from Carla Carter and Dr. Ellen Richie.
We also thank Shawna Johnson and Rebecca Deen for assistance in
preparing the manuscripts, Kent Claypool for providing invaluable
help with flow cytometry analysis, and Nancy Otto and Jimi Lynn
Brandon,oftheScienceParkHistologyCore,forpreparingexcellent
frozen sections and for advising us regarding immunohistochemi-
cal analysis. This work was supported by NIH grants NS43984 and
MH71583 (P.K.Y.), by a Career Re-entry supplement to MH71583
for V.L.S., and by NIH grant MH077470 (V.L.S.). Other support was
provided by NIEHS Center Grant ES07784 and Core Grant CA16672,
both to the M.D. Anderson Cancer Center, Science Park-Research
Division, Smithville, TX, by the Dr. Christian Abee of the Michale E.
Keeling Center for Comparative Medicine and Research, The Uni-
versity of Texas M.D. Anderson Cancer Center, Bastrop, TX, and by
the Longevity Foundation of Austin.
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