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T Regulatory Cells Contribute to the Attenuated Primary CD8+ and CD4+ T

Cell Responses to Herpes Simplex Virus Type 2 in Neonatal Mice

Article in The Journal of Immunology · March 2008

DOI: 10.4049/jimmunol.180.3.1556 · Source: PubMed

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T Regulatory Cells Contribute to the
Attenuated Primary CD8+ and CD4+ T Cell
Responses to Herpes Simplex Virus Type 2 in
Neonatal Mice
Marian A. Fernandez, Franz K. Puttur, Yuan M. Wang,
Wade Howden, Stephen I. Alexander and Cheryl A. Jones
J Immunol 2008; 180:1556-1564; ;
http://www.jimmunol.org/content/180/3/1556

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 The Journal of Immunology

T Regulatory Cells Contribute to the Attenuated Primary

CD8ⴙ and CD4ⴙ T Cell Responses to Herpes Simplex Virus
Type 2 in Neonatal Mice1

Marian A. Fernandez,* Franz K. Puttur,*† Yuan M. Wang,‡ Wade Howden,*†

Stephen I. Alexander,†‡ and Cheryl A. Jones2*†
The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The
neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells
(Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the
draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation
by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but
significant increase in the frequency of CD4ⴙFoxp3ⴙ Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice,

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compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-
specific CD8ⴙ T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice,
and significantly enhanced CD8ⴙ and CD4ⴙ T cell IFN-␥ responses in both infected adults and neonates. Treg depletion also
reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg
suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-␤ in the draining
LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments
support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs. The Journal of
Immunology, 2008, 180: 1556 –1564.

egulatory T cells (Tregs)3 are a population of CD4⫹ T

R
cells that suppress T cell effector responses in vivo (1).
Initially defined by the expression of CD25, the further
identification of the X-linked transcription factor Foxp3 demon-
strated a clear lineage of thymically derived self-Ag-restricted T
cells that limited autoimmune disease (2, 3). Definitive studies of
Treg depletion and reconstitution including neonatal thymectomy
(1, 4), scurfy mice (defective in Foxp3) (5, 6), and humans with
foxp3 mutations and autoimmune disease (IPEX syndrome) (7)
have demonstrated that Tregs dominantly suppress autoimmune
disease from self-reactive T cells. The mechanism of action of
Tregs appears complex involving cell contact, cytokines such as
IL-10 and TGF-␤, and other mechanisms such as CD39 expres-
sion (8). Alternatively, adaptive Tregs can be generated periph-
erally from nonregulatory T cells in the correct environment.
*Centre for Perinatal Infection Research, ‡Centre for Kidney Research, The Chil-
dren’s Hospital at Westmead, Westmead, New South Wales, Australia, and †Disci-
pline of Paediatrics and Child Health, University of Sydney, Sydney, New South
Wales, Australia
Received for publication November 16, 2007. Accepted for publication November
16, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by The Children’s Hospital at Westmead Research Scholar
Award (to C.A.J.).
2
Address correspondence and reprint requests to Dr. Cheryl A. Jones, Centre for
Perinatal Infection Research, The Children’s Hospital at Westmead, Locked Bag
4001, Westmead, New South Wales 2145, Australia. E-mail address: cherylj@chw.
edu.au
3
Abbreviations used in this paper: Treg, T regulatory cell; p.i., postinfection; wt, wild
type; LN, lymph node.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
Foxp3 affects a large number of genes and interacts with NFAT
to establish Treg function (9). A variety of other minor Treg
subtypes have been recently identified including CD8⫹ Tregs
that appear to limit certain CD4⫹ T cell effector populations
(10). Tregs have also been reported to limit excessive adaptive
responses to foreign Ag after infection thereby limiting immu-
nopathology (11, 12), although this can result in persistent in-
fection by some infectious agents and/or limit protective re-
sponses to acute infection (12–15).
Other studies have shown that Tregs modulate antiviral effector
T cell responses in adult humans and adult mice, including to HSV
(15, 16), hepatitis C virus (17, 18), HIV (19, 20), CMV (20), and
EBV (21). However, there is limited data on the role of Tregs in
the modulation of effector responses to infection in infancy or
childhood. Depletion of Tregs from cord blood or postnatal blood
samples has been shown to enhance Ag-specific T cell function in
vitro in infants exposed to malaria (22). Enhanced Treg activity in
HIV-exposed infants has also been associated with reduced verti-
cal transmission of HIV, potentially because Tregs suppressed
CD4⫹ T cell activation, and thereby reduced the potential pool of
targets for virus entry (23). Thus, although Tregs are likely to play
a crucial role in neonatal infections, their role is yet to be fully
defined.
The newborn immune system is skewed toward tolerance
through developmental immaturity, a low number of immune ef-
fectors, and production of Th2 cytokines, although protective im-
munity can be induced under conditions of optimal priming (24).
The newborn adaptive response to some intracellular pathogens,
such as HSV, is weak in magnitude and poorly protective making
them highly susceptible to severe disease (25–28). At 1 wk of age,
the mouse immune system resembles the more mature human
neonate (24). HSV infection in 1-wk-old mice has proven an

www.jimmunol.org
The Journal of Immunology
effective model of the human disease (28, 29). The development
of CD4⫹CD25⫹ Tregs in the thymus and spleen of mice has
been well defined (30 –34). Although Tregs have been shown to
undergo proliferation in peripheral lymph nodes (LN) of neo-
natal mice in an IL-2-dependent manner (33), the development
of naturally occurring Foxp3⫹ Tregs at this site is less well
described.
We have previously reported that HSV induces an attenuated
primary Th1 CD4⫹ and CD8⫹ T cell response in neonatal mice
at day 5 p.i. (28). More recently we observed that the neonatal
murine CD8⫹ T cell effector response to HSV is of slow onset
with a shortened peak response compared with adult controls
(35). Given the critical role of Tregs in regulating effector re-
sponses, we have used a murine model of neonatal HSV type 2
(HSV-2) infection to determine whether dominant CD4⫹
CD25⫹ Treg responses could account for the observed attenu-
ated HSV-specific T cell effector responses in neonates in vivo.
Mice were infected with either wild-type (wt) virus or with a
nonlethal replication-defective HSV-2 strain, dl5–29 (28, 36 –
37), allowing analysis beyond 3 days.
Materials and Methods
Animals, viruses, and inoculations
Animal experiments were conducted with the approval of The Children’s
Hospital at Westmead (Westmead, New South Wales, Australia) and West-
mead Hospital Animal Ethics committees. Female C57BL/6 mice, 4 – 6 wk
of age were purchased from the Animal Resource Centre (Perth, Australia),
acclimatized, and used as controls or used as breeders for experiments.
Neonatal mice were infected at 1 wk of age unless stated otherwise (24).
Adult and neonatal mice were infected with either 1 ⫻ 105 PFU/mouse of
wt HSV-2 stain 186syn⫹-1 (38) or 2 ⫻ 105 PFU/mouse of the replication-
defective HSV-2 186syn⫹-1 mutant virus, dl5–29, (UL5⫺/UL29⫺) (36), or
mice were mock-infected with an equivalent volume of low endotoxin PBS
(Invitrogen Life Technologies) via s.c. inoculation (10 ␮l) into each hind
footpad. Viruses were propagated and stored as previously described (29).
The HSV-2 replication-defective virus dl5–29 and the complementary cell
line V5–29 (36) were provided by D. Knipe (Harvard Medical School,
Boston, MA). Adoptive transfer of splenocytes for the in vivo CTL assay
was by i.v. injection (100 ␮l) into the tail vein of adults and substernally
into the inferior vena cava of neonates.
Depletion or inactivation of Tregs
For in vivo depletion or inactivation of CD4⫹CD25⫹ cells, mice were
injected with purified rat anti-mouse CD25 IgG1 mAb (PC61; BioExpress)
i.p. into adult mice (0.5–1 mg) or 4-day-old mice (100 ␮g) 3 days before
HSV infection. Control mice were given an equivalent dose of a rat IgG
isotype Ab. The efficacy of CD25 depletion/inactivation was confirmed by
flow cytometry using PE-conjugated anti-mouse CD4 and purified anti-
CD25 biotin (7D4) followed by streptavidin-allophycocyanin.
In vivo HSV-specific CTL assay
HSV-specific CD8⫹ T cell cytoxicity was performed as previously de-
scribed (35, 39). Briefly, single cell splenocytes were prepared as CTL
target cells and controls from syngeneic female adult C57BL/6 mice. Tar-
get cells were pulsed with the MHC class I-restricted immunodominant
epitope for C57BL/6 mice from HSV gB498 –505 peptide (SSIEFARL) (1
␮M; Auspep) for 45 min at 37°C then labeled with a high concentration
(2.5 ␮M) of CFSE (CFSEhigh cells). Control cells were left unpulsed and
labeled with a lower concentration (0.25 ␮M) of CFSE (CFSElow cells). An
equal proportion of these cells were adoptively transferred i.v. (1 ⫻ 107
cells/mouse) into HSV-infected adult or neonatal mice or into mock-in-
fected controls at times postinfection (p.i.) as indicated. The draining pop-
liteal LN and spleens were collected, 4 h after transfer from recipient mice,
made into single cell suspensions, and then analyzed by flow cytometry
(data acquisition on FACSCalibur, analysis with CellQuest software; both
from BD Biosciences). The percentage of HSV-specific lysis in vivo was
calculated by comparing differential CFSE intensity according to the following
formulas: HSV gB-specific lysis ⫽ (percentage CFSElow/percentage
CFSEhigh); percentage of specific lysis ⫽ (1 ⫺ (unprimed/primed) ⫻ 100)
(39).
1557
Abs, intracellular cytokine staining, and flow cytometry
Draining popliteal LN cells from HSV-infected neonatal or adult mice or
mock-infected controls were blocked in PBS containing 1% Fc block
(2.4G2), 1% human FBS, 1% normal rat serum (Animal Resource Cen-
tre, Perth, Australia), and 1% normal goat serum (Silenus) for 0.5–1 h
at 4°C. Abs obtained from BD Biosciences were: anti-CD4-PE (GK1.5),
purified anti-CD25-biotin (7D4) (secondary reagent was streptavidin-
allophycocyanin), anti-mouse Foxp3 (FJK-16s), anti-CD8-PerCP (53-
6.7), anti-granzyme B allophycocyanin (MHGBO5), and anti-IFN-␥-
FITC (XMG1.2). Surface staining was at 4°C for 20 min. For
intracellular Foxp3 staining, surface stained cells were then fixed and
permeabilized at 4°C for 18 h, then washed in perm/wash buffer at 4°C
for 30 min, all in the dark. For IFN-␥ and granzyme B intracellular
cytokine staining, cells were first restimulated with gB498 –505 peptide (5 ␮g/
ml) for HSV-specific CD8⫹ T cell cytokine production
or UV-inactivated wt HSV-2 (strain 186, multiplicity of infection
of 5) for CD4⫹ T cell stimulation, at 37°C, 5% CO2 in RPMI 1640 (In-
vitrogen Life Technologies and CALife Technologies) containing 10%
FCS, 2 mM L-glutamine, 2 ⫻ 10⫺2 mM 2-ME (Sigma-Aldrich), and 2%
penicillin-streptomycin. Brefeldin A (GolgiPlug; BD Biosciences) was
added to a 1 ␮g/ml final concentration for the final 4 h of stimulation as
previously published (28). Cells were then fixed, permeabilized, and
stained for intracellular determination of Foxp3. Data were acquired on a
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BD FACSCalibur flow cytometer and analyzed using CellQuest software
(BD Biosciences).
Virus titration
The titer of HSV-2 in the tissues of both adult and neonate mice was
determined by standard plaque assay as reported (29). In brief, the brain,
draining LN, and footpad were collected at days 1, 2, and 3 p.i. and frozen
on dry ice in 0.5 ml of assay medium (PBS containing 0.1% FBS, 0.1%
glucose, and 0.1% CaCl2). Organs were stored at ⫺80°C until assay. For
assay, tissues were then thawed on ice, homogenized using a sterile Kontes
pestle, and plated on Vero cells in duplicate using serial 10-fold dilutions
of 1-ml aliquots per 6-well plates. Infectious titer was determined after
48 h, after fixing the monolayers with methanol, then staining with 1⫻
Giemsa.
RNA isolation, TGF-␤ RT-PCR, and IL-10 real-time PCR
Total RNA was extracted from the draining popliteal LN of mice using
TRIzol reagent (Invitrogen Life Technologies). cDNA was prepared using
a known and constant concentration of total RNA and reverse transcribed
using Superscript II reverse transcriptase (Invitrogen Life Technologies)
and random primer (Promega).
The expression of TGF-␤ was measured by RT-PCR, as previously
published (40). cDNA was subjected to nested PCR amplification using
external and internal primers and ␤-actin as control. The PCR conditions
were 95°C for 5 min (1 cycle); 95°C for 45 s, 60°C for 45 s, and 72°C for
1 min (35 cycles) with the final cycle at 72°C for 7 min. The following
primers were used: TGF-␤ 5⬘-TGGACCGCAACAACGCCATCTATGAG
AAAACC-3⬘ and 5⬘-TGGAGCTGAAGCAATAGTTGGTATCCAGG
GCT-3⬘; and ␤-actin 5⬘-TGGGTCAGAAGGACTCCTATG-3⬘ and 5⬘-CAG
GCAGCTCATAGCTCTTCT-3⬘. The PCR products were run on 2%
agarose gels and visualized under UV light after staining with ethidium
bromide (0.5 ␮g/ml) using Gel-Doc 1000 (Bio-Rad).
Real-time PCR for IL-10 was performed on cDNA obtained from the
draining LN as described. The PCR master mix contained 0.3 ␮M prim-
ers and 0.05 ␮M SYBR Green probe and was cycled at 95°C for 10 min
(1 cycle), 95°C for 40 cycles of 15 s/cycle, and 60°C for 1 min (1
cycle). IL-10 mRNA expression was quantified by real-time PCR using
the ABI PRISM 7700 (PE Applied Biosystems). IL-10 mRNA expres-
sion was calculated by comparative threshold method (or ⌬Ct) to de-
termine the change in IL-10 gene expression in relation to 18 S rRNA,
which was used as internal control. The following primers and probes
were used: 18 S rRNA primer 5⬘-GTAACCCGTTGAACCCCATT-3⬘
and 5⬘-CCATCCAATCGGTAGTAGCG-3⬘ 18 S rRNA probe; IL-10
primer 5⬘-CTGGACAACATACTGCTAACCG-3⬘ and 5⬘-ATTTCCGAT
AAGGCTTGGCAAC-3⬘ IL-10 probe. IL-10 was expressed relative to
the level detected in draining LN of an uninfected age matched sample.
Each sample was analyzed in triplicate.
Statistics
The Student’s two-tailed t test or the Mann-Whitney U test were used
where appropriate to calculate the statistically significant differences be-
tween samples. A value for p ⬍ 0.05 was considered significant.
1558 Tregs SUPPRESS THE PRIMARY NEONATAL T CELL RESPONSES TO HSV
FIGURE 1. Tregs in the peripheral LN of naive neonates reach adult
proportions by day 7 of life. The percentage and number of Tregs in the
popliteal LN and spleens were measured in 1- to 7-day-old C57BL/6 mice
and female adult (6 wk of age) mice (n ⫽ 4 – 6 mice/age/time point). Cells
were analyzed for surface expression of CD4 and CD25, and intracellular
expression of Foxp3 by flow cytometry. Viable cells were gated on lym-
phocytes. Tregs were defined as the percentage of cells that coexpress
CD4⫹, CD25⫹, and Foxp3⫹. The mean percentage ⫾ SEM of Tregs and
of CD4⫹ T cells is shown, as well as the absolute number of Tregs ⫾ SEM
in the draining LN (A) or spleen (B). C, Representative dot plots for LN (A)
and spleen (B) also show the proportion and number of Tregs. Data are
from one of two independent experiments.
Results
Tregs in murine peripheral LN reach adult proportions by
day 7 of life
To study the effect of HSV infection on Treg frequency and function
in neonatal mice at the site of T cell activation, we characterized the
presence of Tregs in peripheral LN and spleen. The transcription
factor Foxp3 is the most consistent marker of naturally occurring
Tregs in humans and mice (9, 41– 44). The percentage and number
of CD4⫹CD25⫹Foxp3⫹ Tregs within the total lymphocyte popu-
lation in the popliteal LN and spleen was therefore determined by
flow cytometry in naive mice on days 1, 3, 5, and 7 of life, and in
adult female mice age 60 days of life (Fig. 1). The percentage of
total CD4⫹ T cells was determined for comparison because CD4⫹
T cell numbers are low in neonatal LN in the first days of life (33).
The proportion of Tregs in the peripheral LN was observed to
mirror the changing proportions of CD4⫹ T cells in newborn mice
over the first week of life. The frequency of Tregs steadily in-
creased in the LN from very low proportions on days 1, 2, and 3
of life, with a larger increase on day 5 of life, to reach the pro-
portion observed in naive adult female mice by day 7 of life
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FIGURE 2. The percentage of Tregs in the draining LN of neonatal and
adult mice after HSV infection. Neonatal (1-wk-old) mice and female adult
controls (n ⫽ 8 –12 mice/age group/intervention/time point) were infected
s.c. with 2 ⫻ 105 PFU/mouse of the HSV-2 mutant dl5–29 (HSV) or
mock-infected with PBS (uninfected). Draining LN cells were collected at
times indicated postinoculation, stained for surface CD4 and CD25 and for
intracellular Foxp3, and analyzed by flow cytometry. A, Mean percent-
age ⫾ SEM of CD25⫹Foxp3⫹ cells after gating on live CD4⫹ T cells from
two experiments is represented. ⴱ, p ⬍ 0.05, by Mann-Whitney U test for
HSV dl5–29 strain-infected mice vs age-matched control (n ⫽ 18 mice) at
day 3 p.i. B, Mean total cell number of Tregs ⫾ SEM is shown. C, Dot plot
representation of adult and neonatal samples from A after gating on CD4⫹
T cells.
(⬃3.5% at days 7 and 60 of life), although the number of Tregs
was still reduced at this time (Fig. 1, A and C). In contrast, con-
sistent with previously published reports (30, 33), the proportion
and number of Tregs in the spleen of newborn mice remained low
in the first week of life, (Fig. 1, B and C). Thus, 1-wk-old mice
have adult proportions of Foxp3⫹ Tregs in the draining LN.
There is an increased frequency of Tregs in the draining LN of
neonatal and adult mice at early times p.i. HSV
The wt HSV-2 strain causes lethal infection even in adult mice
(29). Therefore, to assess the effect of HSV infection on the fre-
quency of CD4⫹Foxp3⫹CD25⫹ Tregs in the LN, female adult and
1-wk-old mice were infected with the nonlethal HSV-2 replica-
tion-defective virus dl5–29 (28, 29), and the percentage of Tregs in
the draining LN was determined by flow cytometry after gating on
CD4⫹ T cells at selected time points on days 1–14 p.i. (Fig. 2, A
and C). HSV infection induced a small, but significant increase in
the percentage of Tregs in the draining LN of infected neonates
and adult mice compared with the percentage found in age-
matched mock-infected controls at day 3 p.i. (neonates: 10.3 ⫾
0.8% vs 8.5 ⫾ 0.4%; adults: 10.8 ⫾ 1.1% vs 9.1 ⫾ 0.3%; p ⬍
0.05, infected vs mock-infected mice by Mann-Whitney U test,
n ⫽ 18 mice). The frequency of Tregs was observed to decrease to
levels observed in age-matched mock controls by day 6 p.i. in
neonates and adults. The number of Tregs peaked in adults and
neonates at day 6 p.i. However, the number of Tregs remained
significantly reduced in HSV-infected neonates compared with in-
fected adults on days 1– 6 (Fig. 2B) ( p ⬍ 0.001, on days 1, 3, 6 p.i.
The Journal of Immunology
FIGURE 3. Treg depletion enhances neonatal HSV-specific CTL re-
sponse and total and CD8⫹ T cell number in draining LN at day 4 p.i.
Four-day-old mice and female adult mice were depleted of Tregs (⫺) by
i.p. injection with anti-CD25 mAb (PC61) to yield ⬎95% depletion in
neonates and ⬎90% in adults by flow cytometry at day 3 postinjection
(data not shown) or injected with an isotype control Ab (Treg ⫹). Three
days later, mice were infected with 2 ⫻ 105 PFU/mouse of the HSV-2
mutant strain dl5–29 (HSV ⫹) or mock-infected with PBS (HSV ⫺) (n ⫽
5–11 mice/intervention/age group). CFSE-labeled HSV gB498 –505 peptide-
primed targets (CFSEhigh) and unprimed controls (CFSElow) were injected
i.v. 4 days p.i. In vivo HSV-specific CTL was measured 4 h posttransfer of
targets. A, Mean percentage ⫾ SEM of HSV-specific CTL lysis is shown.
B, Histograms from A indicating percentage HSV-specific lysis. The effect
of Treg depletion on total cell number (C) and CD8⫹ T cell number (D)
was determined in the draining LN of HSV-infected neonates and adults by
trypan blue exclusion staining and by flow cytometric analysis, respec-
tively. ⴱ, p ⬍ 0.01, Mann-Whitney U test in (n ⫽ 18 mice) A and (n ⫽ 11
mice) C and D. Data are from one of two separate experiments.
in infected adults vs infected neonates, by Mann-Whitney U test,
n ⫽ 18 mice). Thus acute cutaneous HSV infection transiently
increases the proportion of Tregs in the draining LN of neonatal
mice shortly after infection.
Treg depletion significantly enhances neonatal HSV-specific
CD8⫹ T cell cytotoxicity, cell number, and activation
at day 4 p.i.
We have previously observed that the primary CD8⫹ CTL re-
sponse to HSV in neonatal mice is associated with reduced CD8⫹
T cell expansion and short duration of effector activity compared
with adult controls (28, 35). Depletion of Tregs before wt HSV-1
infection of adult mice has been reported to enhance CD8⫹ T cell
expansion, activation, and effector function (16). To assess the
effect of Treg depletion on the primary HSV-specific CTL re-
sponse in neonatal mice (at day 7 of life), 4-day-old mice and adult
controls were injected i.p. with the anti-CD25 Ab (PC61) 3 days
before HSV infection. A separate group of mice were given a rat
IgG isotype Ab as a control. PC61 treatment resulted in a ⬎90%
reduction in the percentage of CD4⫹CD25⫹ cells in adults and
a ⬎95% in neonates by flow cytometry at 3 days postinjection
(i.e., just before HSV infection) (data not shown). Mice were
infected s.c. in the footpad with 2 ⫻ 105 PFU/mouse of HSV-2
strain dl5–29 3 days after depletion or mock-infected with low
endotoxin PBS. The cytolytic capacity of HSV-specific CD8⫹ T
cells was determined in draining LN at day 4 p.i. using an in
vivo CTL assay that assesses the recovery of CFSE-labeled
HSV peptide-pulsed (gB498 –505) targets in the LN injected 4 h
before assay (Fig. 3, A and B) (35, 39). Treg depletion was
observed to significantly increase HSV-specific CD8⫹ T cell
cytotoxicity in neonatal mice at day 4 p.i. ( p ⬍ 0.01, by Mann-
Whitney U test, n ⫽ 18 mice).
1559
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FIGURE 4. Treg depletion enhances CD25 expression by CD8⫹ T cells
after primary HSV-2 infection in neonatal mice. Neonatal (1-wk-old) and
female adult mice were injected i.p. with anti CD25 mAb (PC61) (Treg ⫺)
or with an irrelevant isotype control Ab (Treg ⫹) 3 days before infection
with HSV-2 dl5–29 (HSV ⫹) or mock infection with PBS (HSV ⫺).
Draining LN cells were collected day 4 p.i. (n ⫽ 5– 6 mice/age/interven-
tion) and stained for CD8 and the activation marker CD25, then analyzed
by flow cytometry after gating on viable cells. A, Mean percentage ⫾ SEM
of CD8⫹ T cells that coexpress CD25 is shown. B, Representative dot plots
from A indicate proportion of CD8⫹CD25⫹ T cells expressed. ⴱ, p ⬍ 0.05,
neonates infected vs neonates depleted and infected by Mann-Whitney U
test (n ⫽ 12 mice). Data are from one of four separate experiments.
To test whether this robust increase in HSV-specific CTL re-
sponse after Treg depletion in HSV-infected neonates was due to
enhanced CD8⫹ T cell expansion or recruitment, the total cell
number in the LN and CD8⫹ T cell number was measured by flow
cytometry in the draining LN at day 4 p.i. (Fig. 3, C and D). Both
cell counts were observed to be significantly increased at this site
in Treg-depleted HSV-infected neonatal mice compared with age-
matched HSV-infected controls ( p ⬍ 0.01, Mann-Whitney U test,
n ⫽ 11 mice). Nonsignificant increases in both parameters were
observed in Treg-depleted infected adults ( p ⬎ 0.05, Mann-Whit-
ney U test, n ⫽ 11 mice) (Fig. 3, C and D).
Expression of stable activation markers in HSV-infected
adult mice has been shown to closely correlate with CTL ef-
fector function (45). Therefore CD25 expression on CD8⫹ T
cells from the draining LN was measured at day 4 p.i. (Fig. 4).
In addition to the positive effects on CD8ⴙ T cell cytotoxicity
and number, depletion of Tregs also resulted in an ⬃60% in-
crease in the proportion of CD8⫹ T cells that expressed CD25
in HSV-infected neonatal mice compared with nondepleted in-
fected neonatal mice ( p ⬍ 0.01, Mann-Whitney U test, n ⫽ 11
mice). Treg depletion also induced a nonsignificant increase in
the proportion of CD8⫹CD25⫹ T cells in HSV-infected adult
mice. Thus, Tregs contribute to the suppression of HSV-specific
CD8⫹ cytotoxicity and reduced CD8⫹ T cell expansion or re-
cruitment into the draining LN in neonatal mice.
1560 Tregs SUPPRESS THE PRIMARY NEONATAL T CELL RESPONSES TO HSV
FIGURE 5. Treg depletion increases CD8⫹ and CD4⫹ T cell IFN-␥
responses to primary HSV-2 infection in neonatal and adult mice. Neonatal
(1-wk-old) and adult mice (n ⫽ 6 – 8 mice/age group/time point) were
treated with anti-CD25 mAb (PC61) (Treg ⫺) or irrelevant isotype control
(Treg ⫹) 3 days before inoculation with 2 ⫻ 105 HSV-2 mutant dl5–29
(HSV ⫹) or PBS (HSV ⫺). Draining LN cells were collected day 4 p.i.,
fixed, permeabilized, stained for CD8⫹ or CD4⫹ and for IFN-␥, and ana-
lyzed by flow cytometry. A, Mean percentage ⫾ SEM of CD8⫹ T cells or
CD4⫹ T cells that express IFN-␥ is shown. B, Representative dot plots
from A indicating proportion of cells that express IFN-␥ and either CD8⫹
or CD4⫹. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 by Mann-Whitney U test (n ⫽ 12 mice)
or (n ⫽ 15–17 mice/intervention/age) for CD8⫹ T cells and CD4⫹ T cells,
respectively. Data are from one of two separate experiments with similar
results.
Depletion of Tregs increases neonatal T cell IFN-␥ and
granzyme B expression after acute HSV infection
T cell-derived IFN-␥ production plays a critical role in the immune
response to HSV by limiting spread of infectious virus and acti-
vating other immune effectors (46). Therefore, we tested whether
depletion of Tregs enhanced T cell IFN-␥ expression in the drain-
ing LN after HSV infection, using intracellular cytokine staining
and flow cytometry (Fig. 5). We observed an increase in the per-
centage of CD8⫹ T cells that express IFN-␥ at day 4 p.i. in the
depleted infected neonatal mice compared with nondepleted in-
fected controls (3.6 ⫾ 0.82% vs 1.0 ⫾ 0.27%, p ⬍ 0.01, Mann-
Whitney U test, n ⫽ 12 mice). A 2- to 3-fold increase in IFN-␥⫹
CD8⫹ T cell response was also observed in the depleted infected
adult mice compared with nondepleted controls ( p ⬍ 0.05, Mann-
Whitney U test, n ⫽ 12 mice). Similarly, Treg depletion induced
increased IFN-␥ expression by CD4⫹ T cells at day 4 p.i. in HSV-
infected neonatal mice (5.0 ⫾ 0.18% vs 4.1 ⫾ 0.13%, p ⬍ 0.05,
Mann-Whitney U test, n ⫽ 17 mice) and in adult mice compared
with nondepleted infected controls (6.1 ⫾ 0.5% vs 4.4 ⫾ 0.4%,
p ⬍ 0.05, Mann-Whitney U test, n ⫽ 15 mice). Thus, Treg de-
pletion enhances CD8⫹ and CD4⫹ T cell IFN-␥ responses shortly
after primary HSV infection of neonatal and adult mice.
Granzyme B is one of the cytolytic granules secreted by acti-
vated CD8⫹ T cells for CTL-mediated killing of virus-infected
targets (47). We therefore measured the effect of Treg depletion on
CD8⫹ T cell granzyme B expression after HSV infection. Adult
and neonates were depleted of Tregs before infection with dl5–29,
cells were collected from the draining LN at day 4 p.i. and ana-
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FIGURE 6. Treg depletion increases neonatal CD8⫹ T cell granzyme B
expression after HSV-2 infection. Neonatal (1-wk-old) and adult mice were
treated with anti-CD25 mAb (PC61) (Treg ⫺) or irrelevant isotype control
(Treg ⫹) 3 days before infection with 2 ⫻ 105 HSV-2 dl5–29 (HSV ⫹) or
mock infection with PBS (HSV ⫺) (n ⫽ 5– 6 mice/age group/intervention).
Draining LN cells were collected day 4 p.i., fixed, permeabilized, stained
for CD8 and for intracellular granzyme B, and then analyzed by flow cy-
tometry. A, Mean percentage of granzyme B⫹ CD8⫹ T cells at day 4 p.i.
B, Representative FACS plots show percentage of granzyme B⫹ CD8⫹ T
cells from A. ⴱ, p ⬍ 0.01 by Mann-Whitney U test (n ⫽ 12 mice). Results
shown are from one of two separate experiments with similar results.
lyzed by flow cytometry for the coexpression of CD8⫹ and intra-
cellular granzyme B (Fig. 6). We observed that Treg depletion
significantly enhanced the frequency of granzyme B⫹ CD8⫹ T
cells in neonatal mice after HSV infection compared with age-
matched infected mice without prior Treg depletion (4.7 ⫾ 0.4%
vs 1.6 ⫾ 0.2%, p ⬍ 0.01, Mann-Whitney U test, n ⫽ 12 mice). In
contrast, the expression of CD8⫹ T cell granzyme B in HSV-
infected adult mice was not altered by prior depletion of Tregs.
Thus HSV-induced Tregs suppressed both IFN-␥ expression and
granzyme B expression by neonatal CD8⫹ T cells.
Treg depletion before wt HSV-2 infection reduces the titer of
infectious HSV in the LN and CNS but not the site of infection
Having shown that Treg depletion enhances neonatal CD8⫹ T cell
and CD4⫹ T cell function, we next wanted to define the effect of
Treg depletion on the titer of infectious HSV from the site of
infection, nervous system and organs. For this investigation, neo-
natal and adult mice were infected with a high dose of wt HSV-2.
The site of infection (footpad), LN, and brain were collected at
days 1, 2, and 3 p.i., and the titer of infectious HSV was deter-
mined by standard plaque assay (Fig. 7). Later time points were
not studied due to the rapid onset of lethal encephalitis in HSV-
infected neonatal mice. We observed that Treg depletion did not
significantly alter the titer of infectious HSV in the footpad of
neonatal mice, or at any of the measured sites in adult mice. In
contrast, Treg depletion induced a significant reduction in the titer
The Journal of Immunology
FIGURE 7. Treg depletion reduces the titer of infectious wt HSV-2 from the LN and brain of neonatal mice but not from the site of infection. Neonatal
and adult mice (n ⫽ 3– 8 mice/treatment group/age) were infected s.c in the footpad with 1 ⫻ 105 PFU/ml of wt HSV-2 (strain186). On days 1, 2, and 3 p.i.,
the hind feet, draining LN, and brain were removed and homogenized in virus diluent. The resulting supernatant was used in a Vero cell-based viral plaque
assay to determine the virus titer. Mean titer (log10 PFU/ml) ⫾ SD per organ is shown, from one of two separate experiments with similar results. ⴱ, p ⬍
0.001; ⴱⴱ, p ⬍ 0.0001, infected vs depleted infected, determined by two-tailed Student’s t test. The level of detection of infectious virus is indicated (broken
horizontal line).
of infectious virus in both the LN and brain of neonatal mice
compared with nondepleted virus-infected controls at days 2 and
3 p.i. ( p ⬍ 0.0001, days 2 and 3 p.i. in LN and day 3 p.i. in brain;
p ⬍ 0.001 day 2 p.i. in brain, by Student’s two-tail t test). Devel-
opment of lethal HSV disease was not used as an endpoint of the
study for ethical reasons. However, despite the reduction in virus
titer, signs of lethal encephalitis were observed in both Treg-de-
pleted and nondepleted infected neonates by day 3 p.i. Thus, de-
pletion of Tregs in neonatal mice improves host responses against
infectious HSV systemically and in the brain.
HSV induces increased expression of TGF-␤ but not IL-10 in
the draining LN of neonatal mice
The role of immunosuppressive cytokines (IL-10, TGF-␤) in the
suppression of CD8⫹ CTL effector activity by Tregs was also eval-
uated at day 4 p.i. with the replication-defective HSV-2 mutant
dl5–29. Neonatal mice showed increased TGF-␤ expression in the
draining popliteal LN by RT-PCR compared with uninfected ne-
onates (Fig. 8A). There was also a smaller increase in TGF-␤ ex-
FIGURE 8. HSV infection increases neonatal TGF-␤ but not IL-10 ex-
pression in the draining LN shortly after infection. Neonatal and adult mice
were inoculated s.c. with HSV-2 dl5–29 (HSV ⫹) or with PBS (HSV ⫺).
Draining LN cells were collected day 4 p.i., pooled (n ⫽ 6 mice/age group/
intervention), and subjected to RT-PCR to determine the expression of
TGF–␤ (A) or subjected to real-time PCR to determine the expression of
IL-10 (B). A, Photograph of RT-PCR products of TGF-␤ and ␤-actin re-
solved on an agarose gel and photographed under UV light. B, Mean value
of IL-10 ⫾ SEM in whole LN homogenates of dl5–29-infected mice rel-
ative to the expression in age-matched uninfected controls is shown. Data
are from one of three separate experiments with similar results. ⴱ, p ⬍ 0.05
infected vs uninfected, determined by two-tailed Students’ t test.
1561
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pression after HSV infection in adult mice compared with age- and
sex-matched mock-infected controls. In contrast, IL-10 expression
determined by real-time PCR was significantly increased in the
draining LN of infected adults alone ( p ⬍ 0.05, Student’s two-
tailed t test) (Fig. 8B). Thus, Treg suppression is associated with
enhanced TGF-␤, but not IL-10 expression in the draining LN of
neonatal mice after HSV infection.
Discussion
HSV causes severe disease in the newborn period associated with
reduced primary and memory adaptive response (48). Given the
critical role of Tregs in modulating effector responses, we inves-
tigated whether Tregs suppress the primary T cell responses to
HSV in neonatal mice. We observed an increased frequency of
Tregs in the draining LN of neonatal mice shortly after HSV in-
fection, associated with increased expression of Foxp3 at this site.
Furthermore, depletion of Tregs from neonatal mice before HSV
infection significantly enhanced the magnitude of HSV-specific
CD8⫹ T cell effector function, activation, and cell number and
CD4⫹ T cell IFN-␥ expression more profoundly than depletion
from adult controls, and decreased the titer of infectious virus from
the periphery and nervous system. HSV was also observed to in-
crease the expression of TGF-␤ but not IL-10 in neonatal LN. To-
gether these data suggest that Tregs suppress neonatal T cell effector
responses to acute viral infection, which may contribute to increased
virulence of intracellular pathogens in the newborn period.
Age affects both the number and function of Tregs in humans
and mice (10, 30 –34, 49 –51). The murine thymus contains a low
frequency of naturally occurring Tregs at birth that increases sig-
nificantly over the first week of life to approach adult levels by 3
wk of age in the thymus (9) and 6 wk of age in the spleen (49).
Foxp3 expressing CD4⫹CD25⫹ splenocytes can be detected in
3-day-old mice, but at levels considerably lower than levels in
adult mice (32). The human fetus and newborn display similar
patterns of Treg development at these sites (50), although the fre-
quency of CD4⫹CD25⫹ Tregs in human cord blood approximates
the proportion found in the blood of healthy adults (51).
CD4⫹CD25⫹ Tregs are present in the LN of 6-day-old mice in
approximate adult proportions (33). However the ontogeny of
Foxp3-expressing Tregs in the murine LN in the first week of life
remains poorly defined. This study demonstrates that Foxp3-ex-
pressing Tregs are first present in detectable levels in the periph-
eral lymphoid tissue of newborn mice from day 3 of life, and reach
1562 Tregs SUPPRESS THE PRIMARY NEONATAL T CELL RESPONSES TO HSV
adult proportions at ⬃day 7 of life. The low number of Tregs in the
LN on days 1–3 of life could be due to the presence of a large
number of precursor cells or a limitation in detection due to small
size of newborn LN and the very low number of T cells.
Having established that the proportion of Tregs in the peripheral
LN of 1-wk-old mice was similar to adults, we next compared the
effect of HSV on the proportion of Tregs at that site. We show that
HSV induces a small but significant increase in the proportion of
Tregs in the draining LN of neonatal mice shortly after infection.
A number of microbial pathogens including viruses have been pre-
viously shown to increase the frequency of Tregs in infected tis-
sues or lymphoid organs of adult mice and humans (12, 52–56).
CD4⫹CD25⫹ Tregs have been shown to suppress CD8⫹ T effector
function and activation to viruses including HSV and to viral vac-
cines in adult humans ex vivo, and in adult mice in vitro and in
vivo (12, 20, 53– 60). Enhanced Treg responses have been detected
in the cord blood of human neonates born to women with Plas-
modium falciparum infection compared with unexposed infants or
infants born to mothers who had been treated for malaria (22).
Increased proportions of Tregs with weak HIV-1 specific T cell
responses have also been detected in the blood of HIV-1-exposed
uninfected infants, compared with unexposed controls (23), sug-
gesting that persistence of in utero Treg responses have suppressed
postnatal HIV-specific effector T cell responses. The report spec-
ulated that the enhanced Treg response may have been beneficial,
protecting infants against vertical infection by reducing T cell ac-
tivation and thereby lower the pool of activated CD4⫹ targets and
hence HIV-1 replication.
In this study, we show that depletion of Tregs before HSV in-
fection significantly increased neonatal HSV-specific CD8⫹ T cell
cytotoxicity, suggesting that enhanced Treg responses to HSV in
the draining LN contribute to the attenuated neonatal CTL cell
response to HSV that we have previously observed (29, 35). Tregs
have been previously shown to suppress HSV-specific CD8⫹ and
CD4⫹ T cell responses to wt HSV-1 (16) and to HSV DNA vac-
cines (59) in adult mice and to wt HSV-2 in humans (61). In this
study, we did not observe enhanced HSV-specific CTL lysis after
Treg depletion in adult mice. The likely explanation for the dis-
crepancy is the timing of the analyses. Our experiments were per-
formed day 4 p.i. when HSV-specific lysis is close to the peak
response in adults (i.e., at ⬃70%), thus limiting detection of minor
augmentation of CTL responses by Treg depletion. The study by
Suvas et al. (16) analyzed adult CTL responses at day 7 p.i. when
the CTL response in HSV-infected adults has waned to 10%.
When we perform our analyses at day 7 p.i., we find a similar
augmentation of adult HSV-specific CTL activity after Treg de-
pletion, in addition to a smaller but significant augmentation in
neonates (data not shown).
Treg depletion before HSV-2 infection enhanced CD4⫹ and
CD8⫹ T cell IFN-␥ expression in adult mice, with similar sized
increases observed in neonatal mice, consistent with previous re-
ports of a variety of viral agents after functional blockade or Treg
depletion in adult mice (12, 16, 58). Tregs have been shown to
suppress granzyme B expression by Friend retrovirus-stimulated
CD8⫹ T cells in vitro (62). We show that Treg depletion signifi-
cantly increased granzyme B expression in HSV-infected neonatal
mice alone. The reason for the age-associated difference is not
clear. Acquisition of granzyme B expression by CD8⫹ T cells after
influenza infection has been tightly associated with cell division
(63). Thus, it is possible that the age-associated augmentation of
CD8⫹ T cell granzyme B expression was related to the larger
increase in CD8⫹ T cell number induced by Treg depletion in
infected neonates.
Treg depletion was also associated with a reduced titer of in-
fectious HSV in the LN and brain shortly after infection in neo-
nates, suggesting that suppression of neonatal immune effectors by
Tregs may contribute to the enhanced virulence of this virus in
newborn animals and humans. Future studies will determine
whether this represents delayed dissemination of infectious virus
to these sites or enhanced clearance by the innate and adaptive
effectors.
We used PC61 mAb to reduce the frequency of naturally oc-
curring Tregs for our functional studies on HSV immunobiology.
Recent reports have indicated that PC61 depletes only a subset of
Tregs (particularly those that are CD69lowFoxp3low) and function-
ally inactivates the remaining naturally occurring Tregs (64, 65).
These studies have also shown that although PC61 treatment sig-
nificantly lowers the CD4⫹CD25⫹ population, it results in only a
small change in the number of CD4⫹Foxp3⫹ cells. In this study
we report ⬎90% reduction in the number of CD4⫹CD25⫹ Tregs
in adult mice treated with high-dose PC61 (500 ␮g/adult mouse
Ab). Furthermore, we observed a 50% reduction in Foxp3 expres-
sion (by real-time PCR) in the LN of mock-infected PC61-treated
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mice (data not shown), consistent with these studies.
Treg suppression of T cell effectors has been reported to occur
by both cytokine-dependent (IL-10 and TGF-␤) and cell contact-
dependent mechanisms (66 – 69). Naturally occurring Tregs in the
conjunctiva of rabbits after ocular HSV-1 infection have been
shown to suppress T cell effectors by cell contact, and not by the
production of suppressive cytokines (69). The mechanisms by
which Tregs suppress T cell effectors after cutaneous HSV infec-
tion have not been fully defined. In this study we show that there
is increased expression of TGF-␤, but not IL-10, in the neonatal
murine LN shortly after cutaneous HSV infection. The neonatal
cytokine microenvironment is generally thought to be Th2-biased
(24). We were therefore surprised that the reduced neonatal CTL
response to HSV was not associated with a greater increase in the
neonatal IL-10 response. However, this observation is consistent
with previous observations by our group and by other groups that
there is a general paucity of both Th1 and Th2 cytokine production
by T cells compared with infected adult controls (27, 28, 70).
TGF-␤ has been reported to exert an inhibitory effect on T cell
differentiation and proliferation. It has also been shown to be re-
quired for the conversion of CD4⫹CD25⫹ T cells into Tregs in
vitro, and to be necessary for the maintenance of Tregs in the
periphery (71). Whether the enhanced expression of TGF-␤ in
HSV-infected neonates directly contributes to the impaired HSV-
specific CTL response and whether Tregs are the source of the
TGF-␤ is yet to be determined.
Tregs have also been reported to have bidirectional interactions
with dendritic cells (72, 73). Neonatal dendritic cells are function-
ally immature compared with adult controls (74). Future work will
therefore also determine the role of age-associated differences in
the interaction of Tregs and dendritic cells in the generation of
antiviral responses. Demonstration of the mechanisms by which
Tregs expand locally in the neonate and suppress Ag-induced ef-
fector responses in the newborn period may give insight into en-
hanced therapeutic strategies while limiting potential autoimmune
side effects.
Acknowledgments
We thank Eddy Hassan, Christine Victoire, Ingrid Evans, Mary Sartor, and
Sanda Lum for technical assistance. We also thank Megan Cameron for
dedicated animal care and Nick Taylor for assistance with the graphics.
Disclosures
The authors have no financial conflict of interest.
The Journal of Immunology
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