Rathod

Suppressive activity and altered conventional phenotype
markers/mediators of regulatory T cells in patients with
self-limiting hepatitis E
S. B. Rathod, R. Das, S. Thanapati, V. A. Arankalle and A. S. Tripathy Hepatitis Group, National
Institute of Virology, Pune, India
Received October 2012; accepted for publication June 2013
SUMMARY. Hepatitis E virus (HEV) is a major cause of self-
limiting acute viral hepatitis in several developing coun-
tries. Elevated levels of peripheral CD4+
CD25+
Foxp3+
,
CD4+
CD25À
Foxp3+
and rise in IL-10 in hepatitis E have
been associated with the involvement of regulatory T cells
(Treg). The functional role of the same is yet elusive. In
the current study, we have assessed (i) Foxp3 expression
by real-time PCR and by flow cytometry, (ii) the levels of
antigen-specific IL-10 and TGF-b by ELISA, (iii) functional
analysis of Treg cells and (iv) expression of Treg-associated
conventional phenotypes by flow cytometry in 54 acute
patients, 44 recovered individuals from hepatitis E and in
33 healthy controls. Foxp3 mRNA elevation in the acute
compared with recovered group and elevation in Foxp3+
cells in both patient groups were significantly elevated. The
levels of IL-10 and TGF-b in the acute patients and TGF-b
in the recovered individuals were elevated. Significantly
higher expression of CTLA-4, PD1, GITR, CD95, CD103
and CD73 on Treg and T effector (Teff) cells was detected
in the patient groups. Treg cells of acute patients and
recovered individuals exhibited suppressive activity indicat-
ing that the Treg cells of hepatitis E patients are functional.
The suppressive capacity of Treg cells in acute hepatitis E
patients was significantly higher compared with the recov-
ered individuals. Based on our findings, the suppressive
functionality of these key markers associated with hepatitis
E Treg function need further exploration to get a better
understanding of the mechanisms of Treg-mediated sup-
pression.
Keywords: CFSE, Conventional phenotypes markers, Foxp3-
mRNA, Functional Treg cells, Teff cells.
INTRODUCTION
Hepatitis E virus (HEV) causes large-scale epidemics and
sporadic cases of acute viral hepatitis in developing coun-
tries [1,2]. Even though, not much is known about the
nature of cell mediated immune responses during infection
with HEV, the literature suggests a definitive role of T cells
in the immunopathogenesis of HEV infection [3]. The ele-
vation of the pro-inflammatory cytokine TNF-a and anti-
inflammatory cytokine IL-10 in the acute patients has been
associated with immunosuppressive immune responses in
hepatitis E [4]. Regulatory T cells (Treg) might be mediat-
ing their suppressive activity through the secretion of anti-
inflammatory cytokines like IL-10 [5]. This goes in parallel
with our previous report of increased IL-10 production in
FHF-E patients irrespective of their outcome [6]. Treg cells
play an important role in the suppression of virus-specific
immune responses [7–9]. The molecular mechanisms by
which Treg cells mediate their suppressive activity remain
poorly understood. Phenotypically and functionally distinct
subsets of Treg cells suppress the proliferation of T effector
(Teff) cells and also inhibit the activation of autoreactive T
cells through the expression of coinhibitory molecules
(CTLA-4, GITR) and production of suppressive cytokines
like IL-10 and TGF-b [10,11]. Surface molecules linked to
Treg cell suppression include PD1/PD1-L [12,13], chemo-
kines/homing receptors, integrins, CD39 and CD73 [14].
Meager information is available regarding the functions of
Tregs in acute infections. In an attempt to identify the key
molecules involved in the Treg-mediated pathogenesis in
self-resolving hepatitis E infection, in the current study, we
have investigated (i) the expression of Foxp3 at the (a)
mRNA level by real-time PCR and (b) protein level by flow
Abbreviations: CFSE, carboxyfluorescein succinimidyl ester;
CTLA-4, cytotoxic T-lymphocyte-associated protein 4; FHF, fulmin-
ant hepatic failure; GITR, glucocorticoid-induced TNFR related
protein; HEV, hepatitis E virus; PBMCs, peripheral blood mononu-
clear cells; PD1L, PD-1 ligand; PD-1, programmed death 1; TGF-b,
transforming growth factor-b.
Correspondence: Anuradha S. Tripathy, Scientist C, Hepatitis
Group, National Institute of Virology, 130/1, Sus Road, Pashan,
411021 Pune, Maharashtra, India.
E-mail: anuradhastripathy@hotmail.com
© 2013 John Wiley & Sons Ltd
Journal of Viral Hepatitis, 2014, 21, 141–151 doi:10.1111/jvh.12125

cytometry, (ii) hepatitis E antigen-specific IL-10, TGF-b lev-
els by ELISA, (iii) the functional analysis of Treg cells and
(iv) expression/involvement of certain key molecules that
are frequently associated with human Treg function (apop-
totic, inhibitory, costimulatory, activation, integrin family
member, adenosinergic machinery and chemokines/hom-
ing receptors molecules) on the gated populations of
CD25+
(Treg) and CD25À
(Teff) cells by flow cytometry in
hepatitis E patients during the acute phase of illness, after
recovery and in healthy controls.
MATERIALS AND METHODS
Study subjects
A total of 131 individuals were included in the study
(Table 1). The patients were enrolled during February
2011–August 2012 and were from two states of Western
India, and the controls were healthy volunteers from the
same population. Diagnosis of hepatitis E was based on the
presence of IgM antibodies to hepatitis E virus (IgM anti--
HEV) as detected by ELISA [15]. The patients were classi-
fied as acute based on the standard clinical and
biochemical criteria [6]. Briefly, AVH-E patients were
defined as those presenting with icterus, dark-coloured
urine, elevated alanine aminotransferase (ALT) (>40 IU/
mL) and/or bilirubin levels (>1 mg/mL) in their serum
and/or presence of bile salts and pigments in the urine
were considered. At enrolment, the patients were con-
firmed to be positive for both serum anti-HEV IgM and
ALT >40 IU/mL but were negative for other viral infec-
tions, including HBV, HCV and human immunodeficiency
virus. The recovered individuals had a previous history of
acute hepatitis E (AVH-E). Control groups were negative
for HBsAg, anti-HCV, IgM/IgG anti-HEV, IgM anti-HAV
antibodies and being of the same age as the patient groups.
None of the patients had a past history of chronic liver dis-
ease and severe systemic illness or was undergoing therapy
at the time of sampling. This study was approved by the
Institutional Ethical Committee for Humans and written
informed consent was obtained from all the participants.
Serological assays
The serological assays and end points of serum ALT, IgM
and IgG levels were determined as previously described [16].
Enumeration of T lymphocytes and Treg cells
Freshly isolated PBMCs (1 9 105
) were surface stained with
APC-labelled anti-human CD3, PE-Cy7-labelled CD4 and
FITC-labelled CD8 with appropriate isotype controls, accord-
ing to the manufacturer’s instructions (BD biosciences, San
Jose, CA, USA). For CD8 Treg, (1 9 105
) PBMCs were sur-
face stained with FITC-labelled CD8 and APC-labelled CD25
surface markers and were then lysed, permeabilized and
stained using anti-human Foxp3 (PE) antibody, along with
appropriate rat IgG2a isotype controls (eBiosciences, San
Diego, CA, USA) [4].
Isolation of Treg and Teff cells
A CD4+
CD25+
CD127dim/À
Regulatory T cell Isolation Kit
(Miltenyi Biotec, Bergisch Gladbach, Germany) was used to
isolate Treg cells and Teff cells according to the manufac-
turer’s instructions. Briefly, non-CD4+
and CD127high
cells
were magnetically labelled with a cocktail of biotin-conju-
gated antibodies and antibiotin microbeads and were sub-
sequently depleted by negative selection. Pre-enriched
CD4+
T cells were then labelled with anti-CD25 microbe-
ads. Subsequently, Treg cells were isolated by positive
selection, and CD4+
CD25À
cells that were not retained in
the magnetic separation column during purification of
CD25+
cells were used as Teff cells. The purity of the iso-
lated Treg cells was confirmed by flow cytometry.
CFSE-based proliferation assays
To assess the suppressive capacity of peripheral
CD4+
CD25+
CD127dim/À
Treg from blood samples, we per-
formed in vitro proliferation assays, in which effector T cells
were stimulated in the absence or presence of Treg. Carboxy-
fluorescein succinimidyl ester (CFSE)-based (Invitrogen, Life
Table. 1 Clinical and virological characteristics of patients and controls, expressed as median (range).
Parameters AVH-E Recovered Controls
Study population n = 54 n = 44 n = 33
Age (Years) 25 (15–59) 33 (17–60) 25 (20–32)
Sex ratio (M:F) 23:31 22:15 21:11
ALT IU/L 356 (53–1700) 26 (15–40) 19 (9–35)
IgM titre 6400 (400–51 200) 1600 (400–25 600) NA
IgG titre 25 600 (400–102 400) 51 200 (800–102 400) NA
Post onset days (POD) 6 (1–31) 90 (80–105) NA
NA, not applicable.
142 S. B. Rathod et al.

Technologies, Carlsbad, CA, USA) labelling and suppression/
proliferation were carried out in 5 AVH-E patients, 4 recov-
ered individuals and 3 healthy controls. Following the CFSE-
labelling method [17], isolated Teff cells were labelled with
5 lM CFSE for 1 9 106
cells/mL. 1 9 104
and 4 9 104
CD4+
CD25À
Teff cells were stimulated with soluble anti-CD3
(5 lg/mL) and anti-CD28 (2 lg/mL) (Miltenyi Biotec). Treg
cells (1 9 104
) were then added to Teff cells (1 9 104
and
4 9 104
) to make 1:1 and 1:4 ratios, respectively. CFSE-
labelled Teff cells without Treg cells served as controls. Sam-
ples were plated in a round-bottom 96-well plate in triplicate
in RPMI 1640 medium (Invitrogen) supplemented with 5% of
foetal calf serum, 2 mmol/L L-Glutamine, 1 mmol/L sodium
pyruvate and 20 lg mL of gentamicin and were kept at
37 °C in 5% CO2. After 3 days, the proliferation of CFSE-
labelled Teff cells was evaluated by flow cytometry as
described elsewhere [18]. Per cent suppression was calculated
using proliferation measurements and the following formula:
suppression (%) = (1ÀTreg:Teff/Teff alone) 9 100.
Foxp3 m-RNA quantification
Total RNA isolated from stored PBMCs in RNA later (Ambi-
on, Life Technologies, Foster City, CA, USA) was quanti-
tated, and cDNA was synthesized from 2 lg of total RNA
using the high-capacity cDNA synthesis kit (Life technolo-
gies, USA). Real-time PCR was performed on an ABI Prism
7300 sequence detection system using cDNA-specific FAM-
MGB–labelled Taqman primer/probe sets for Foxp3
(Hs00203958-m1) [19]. FAM-MGB–labelled GAPDH
(Hs99999905-m1) (Life technologies, Carlsbad, CA, USA)
was used as endogenous control for relative amount of
mRNA in each sample. The efficiencies of the target (Foxp3)
and the reference (GAPDH) were approximately equal, as
assessed in individual validation experiments. Quantities of
target gene expression in the test samples were normalized
to the corresponding GAPDH mRNA level of controls.
Antigen-specific immunoregulatory cytokines
Limitation of the culture supernatant in the suppression
assay led us to analyse the levels of cytokines produced by
PBMCs from patients after in vitro stimulation with HEV-
rORF2 antigen. PBMCs of patients and controls (106
cells/
mL) were cultured with HPLC purified rORF2p (conc.
10 lg/mL) from genotype-1 HEV. Unstimulated PBMCs
served as controls. After 72 h, the culture supernatants
were harvested and were assessed for cytokines in dupli-
cate for each sample. The detection of IL-10 and TGF-b in
all subjects was carried out using standard ELISA kits
(R&D Systems, Minneapolis, MN, USA).
Flow cytometry analysis of surface/intracellular molecules
Monoclonal antibodies used specific for CD4 labelled with
FITC, CD25-APC and PE labelled-CD95 (Fas), CD152
(CTLA-4), CD279 (PD1), CD274 (PD1-L),CD103 (aEb7),
CD194 (CCR4), CD197 (CCR7), CD62L, CD39, CD73,
CD71, HLA-DR, CD69, CD45RA, CD278 (ICOS), CD40,
CD40L (CD154), CD137 (4-1BB), CD70, CD28 (BD bio-
sciences) and CD357 (GITR) (Miltenyi Biotec) with the
appropriate isotype control antibody markers were studied
in the context with the expression on gated (i) CD25+
(Treg) and (ii) CD25À
(Teff) cells of CD4+
T cells in all
studied subjects. The parent percentages from gated lym-
phocytes were calculated on the FACS AriaTM
II instrument
and data analysed by using FACS Diva software (BD bio-
sciences).
Statistical analysis
Data are presented as mean Æ SD unless stated otherwise.
The nonparametric Mann–Whitney U-test was used to
compare data between patients and controls. The differ-
ences were considered statistically significant at two-sided
P-values ≤0.05. Correlation analysis was performed using
Spearman’s rank correlation coefficient. All statistical anal-
yses were performed with ‘SPSS11.0’ software (SPSS Inc.,
Chicago, IL, USA).
RESULTS
Clinical and virological characteristics of the study
subjects
The ALT levels were significantly higher in AVH-E vs
recovered, P = 0.003 and control individuals, P = 0.0001.
The IgM levels were significantly higher in AVH-E vs
recovered, P = 0.01,and the reverse was true in case of
the IgG levels, P = 0.02 (Table 1).
T lymphocyte and Treg cell frequencies
The parent percentages of CD3+
T cells were elevated in
both the patient groups. CD3+
CD4+
T cells were elevated
only in AVH-E vs the control group. Among the patient
categories, the percentage of CD3+
CD4+
T cells were more
in the AVH-E patients, and CD3+
CD8+
T cells were more
in the recovered individuals. The percentages of Foxp3
expression on CD8+
CD25+
, CD8+
CD25À
cells in both
patient groups were significantly elevated vs controls
(Table 2).
Foxp3 expression by flow cytometry and real-time PCR
The percentage of total Foxp3 expression was significantly
elevated in both the patient groups vs controls
(23.72 Æ 1.4, 11.43 Æ 2.7 vs 3.88 Æ 0.56, P = 0.0001
& P = 0.01, respectively). Among patient categories, it was
significantly more in AVH-E (23.72 Æ 1.4 vs 11.43 Æ 2.7
P = 0.002) (Fig. 1a).
Functional regulatory T cells in hepatitis E 143

Due to the constraint of quantity of blood samples from
the patients, Foxp3 mRNA expression was measured from
total PBMCs and was significantly elevated in AVH-E vs
the recovered group (4.24 Æ 1.63 vs 1.03 Æ 0.69,
P = 0.001) after normalization with endogenous control
(Fig. 1b).
Immunoregulatory cytokines
The levels of IL-10 and TGF-b in AVH-E [1.38 (0.36–
1.71), 1.66 (1.10–2.03)] and TGF-b in the recovered
group [1.36 (0.52–1.83)] were elevated vs controls [0.89
(0.19–1.55), 0.94 (0.24–1.41)], respectively. Among the
Table 2 Percentage of parent population of different T-cell subtype frequency
% of cell AVH-E(n = 54) Recovered(n = 20) Control(n = 20) P-valueA
P-valueB
P-valueC
% CD3 62.9 Æ 1.5 66.2 Æ 2.1 58.2 Æ 1.6 NS 0.05 0.005
% CD3+
CD4+
52.5 Æ 0.8 40.8 Æ 2.5 40.3 Æ 2.2 0.0002 0.00001 NS
% CD3+
CD8+
27.1 Æ 1.7 30.6 Æ 1.2 26.2 Æ 0.6 0.03 NS NS
% CD4+
CD25+
4.9 Æ 0.2 3.9 Æ 0.3 2.5 Æ 0.2 0.003 0.002 0.04
% CD4+
CD25À
34.6 Æ 0.7 31.7 Æ 0.8 30.9 Æ 1.1 NS 0.04 NS
% total Foxp3+ 23.72 Æ 1.4 11.43 Æ 2.7 3.88 Æ 0.5 0.002 0.0001 0.01
% Foxp3+
CD8+
CD25+
6.1 Æ 0.8 3.3 Æ 0.7 1.7 Æ 0.3 0.01 0.0001 NS
% Foxp3+
CD8+
CD25À
4.5 Æ 0.5 1.1 Æ 0.3 0.9 Æ 0.4 0.02 0.002 NS
Data are represented as per cent of gated population and are expressed as mean Æ SD. Statistical significance was analysed
by Mann–Whitney U-test. P-valueA
= AVH-E vs recovered; P-valueB
= AVH-E vs control and P-valueC
= Recovered vs con-
trol NS; not significant.
Fig. 1 Foxp3 expression and immunoregulatory cytokines expression in Hepatitis E: (a) Percentage expression of Foxp3
protein on PBMCs shown in box plot. (b) Quantitative expression of Foxp3 mRNA by real-time PCR. (c, d) Hepatitis E
specific TGF-b and IL-10 quantification in the supernatant of recombinant HEV ORF2 protein induced PBMCs, expressed in
median Log10 (range). n, number of patients. Data are representative of mean Æ SD, and P-values were calculated using
Mann–Whitney U-test.

patient categories, the levels of both IL-10 and TGF-b were
elevated in AVH-E (Fig. 1c,d). Values are expressed in med-
ian Log10 (range).
CD4+
CD25+
CD127À/dim
Tregs cocultured with
CD4+
CD25À
effector T cells have higher suppressive
function in AVH-E
An outline of the suppression assay protocol is shown in
Fig. 2a. Teff cells were stimulated with antibodies to CD3
and CD28, and proliferation of the CFSE-labelled Teff cells
was quantified by flow cytometry. The effector T cells in
the presence of Treg at 1:1 and 1:4 ratios resulted in sig-
nificant suppression of proliferation in AVH-E
[45.72 Æ 5.92 (Teff) vs 24.32 Æ 4.17 (Teff+Treg),
P = 0.01] and [45.72 Æ 5.92 (Teff) vs 27.72 Æ 7.39
(Teff+Treg), P = 0.051, respectively]. Similarly, there was
significant reduction in the effector cell proliferation in the
recovered and control individuals [50.16 Æ 6.17 (Teff) vs
25.90 Æ 4.90 (Teff+Treg), P = 0.051]; [50.16 Æ 6.17
(Teff) vs 37.86 Æ 6.13 (Teff+Treg), P = 0.02] and controls
[54.52 Æ 2.23 (Teff) vs 33.97 Æ 5.01 (Teff+Treg),
P = 0.050]; [54.52 Æ 2.23 (Teff) vs 40.93 Æ 0.93 (Teff+-
Treg), P = 0.050], respectively (Fig. 2b–d). As control, the
proliferation of Teff cells in the absence of Treg was
assessed. The presence of Treg cells at a 1:1 ratio resulted
Fig. 2 Effector T-cell proliferation and autologous Treg cell suppressor function in Hepatitis E: CD4+
CD25+
CD127dim/À
regulatory T cells (Treg) and CD4+
CD25À
effector T cells (Teff) were isolated from peripheral blood of 9 patients (5 AVH-E,
4 recovered) and 3 healthy controls. (a) Outline of strategy for performing in vitro suppression assays to measure Treg
functionality. Proliferation was measured by CFSE-labelled Teff cell division by flow cytometry, expressed as mean
percentage proliferation. (b) Treg cell–mediated suppression assays were conducted by culturing 1 9 104
CD4+
CD25+
CD127dim/À
Treg cells and 1:1 to 1:4 Teff cells (1 9 104
and 4 9 104
). For individual group comparisons of
Treg suppressor function, the relative proliferation of Teff cells in the presence of Treg is displayed, indicating that the Treg
from AVH-E patients, recovered and control individuals significantly suppressed the proliferation of Teff cells. (c, d)
Comparison of autologous suppression of patient groups and control individuals at 1:1 and 1:4 Treg to Teff cell ratios. The
data represent a compilation of data from 5 AVH-E patients, 4 Recovered individuals and 3 control individuals performed
in triplicate. Data are expressed as mean Æ SD, and P-values were calculated using Mann–Whitney U-test.

in significant reduction in effector cell proliferation in the
AVH-E group compared with the recovered [39.53% Æ
5.78 vs 24.73 Æ 4.60, P = 0.014] and control groups
[39.53% Æ 5.78 vs 24.87% Æ 1.99, P = 0.025].
There was no significant difference in the percentage of
suppressive activity of Treg cells in the AVH-E patients
based on post onset days of illness (POD) (Table 3).
Flow cytometry gating strategy and analysis
Characterization of Treg was carried out with gating the
lymphocytes on FSC vs SSC dot plot. The T-lymphocyte
subpopulations were further selected on anti-CD4 vs anti-
CD25 dot plots. The percentage of cells expressing different
conventional markers were analysed on CD4+
T cells, con-
sidering two different gates, according to the level of
expression (or not) of CD25 (Fig. 3).
AVH-E patients and recovered individuals exhibit
different levels of CD4+
CD25+
and CD4+
CD25À
Teff
cells
To characterize Treg population, CD4+
vs CD25 dot plots
were carried out and CD25 lymphocytes were classified as
CD25+
and CD25À
T cells (Fig. 3a). Elevated frequencies of
CD4+
CD25+
(Treg) (Fig. 3b) were found in both the patient
groups vs controls, P = 0.002 and P = 0.043, respectively.
The frequencies of CD4+
CD25À
(Teff) cells were elevated
only in AVH-E vs controls, P = 0.02 (Fig. 3c) and compari-
sons among the patient categories revealed the frequencies
of CD4+
CD25+
to be high in AVH-E patients, P = 0.003 as
represented in Table 2.
Expression of conventional markers on CD4+
CD25+
and
CD4+
CD25À
T cells
Expression of inhibitory markers CD95, PD1, PD1L, CTLA-4
and GITR, integrin CD103, homing receptors CCR7, CCR4,
CD62L, ectonucleotidase CD73, CD39, activation markers
HLA-DR, CD71, CD69, na€ıve CD45RA and costimulatory
molecules, ICOS, CD40, CD154, 4-1BB, CD70 and CD28
conventional markers on Treg and Teff cells was addressed
(Fig. 3d–q).
Treg cells showed (i) AVH-E vs control: significantly
higher frequencies of CD95, PD1, PD1L, CTLA-4, GITR,
CD103, CCR7, CCR4, CD73, CD39, HLA-DR, CD71, ICOS,
CD40 and lower frequencies of CD62L, CD28, CD45RA in
AVH-E group; (ii) recovered vs control: significantly higher
frequencies of CD95, PD1, CTLA-4, GITR, CD103, CCR7,
CCR4, CD73, HLA-DR, CD71, CD69, ICOS, CD40, CD154
and lower frequencies of CD62L, CD39, CD45RA in recov-
ered group; (iii) AVH-E vs recovered: significantly higher
frequencies of CD95, CTLA-4, CCR4, CD62L, CD39, CD73,
CD45RA and lower frequencies of GITR, CD103, CCR7,
HLA-DR, CD71, CD69, CD28, ICOS,CD154 in AVH-E were
observed (Fig. 3d–j).
Teff cells showed (i) AVH-E vs control: significantly
higher frequencies of CD95, PD1, CTLA-4, GITR, CD103,
CCR4, HLA-DR, CD69, CD73, ICOS, CD40, CD154 and
lower expression of CCR7, CD45RA in AVH-E group; (ii)
recovered vs control: significantly higher frequencies of
CD95, PD1, CTLA-4, GITR, CD103, CCR7, HLA-DR, CD71,
CD69, ICOS, CD40, CD154 and lower frequencies of CD62L,
CCR4, CD39, CD45RA and CD28 in recovered group; (iii)
AVH-E vs recovered: significantly higher frequencies of
CTLA-4, GITR, CCR4, CD62L, CD73, CD39, CD45RA and
lower frequencies of CD95, CD103, CCR7, CD71, CD69,
CD28, CD40 in AVH-E were observed (Fig. 3k–q).
Spearman correlation analysis
To investigate whether alteration in surface and intracellu-
lar marker expression on circulating CD4+
CD25+
and
CD4+
CD25À
T cells correlated with the expression of Foxp3
mRNA and protein level, cytokines at the protein level,
serum IgM anti-HEV level, spearman analysis was carried
out.
In AVH-E, expression of CD39 was negatively correlated
with IgM levels on CD4+
CD25+
(r = À0.561, P = 0.051)
and on CD4+
CD25À
(r = À0.739, P = 0.001). TGF-b levels
positively correlated with total Foxp3 (r = 0.56,
P = 0.053).
In the recovered group, expression of CD154 was nega-
tively correlated with IgM levels on CD4+
CD25+
T cells
(r = À0.532, P = 0.016). TGF-b levels positively correlated
with (i) expression of CD103 on Treg and Teff cells
(r = 0.604, P = 0.005 and r = 0.623, P = 0.003, respec-
tively) and (ii) the expression of CCR4 (r = 0.514,
P = 0.031) on CD4+
CD25+
cells only. Foxp3-mRNA level
positively correlated with (i) CD69 expression (r = 0.546,
P = 0.029) on CD4+
CD25+
and (ii) CTLA4 expression
(r = 0.512, P = 0.048) on CD4+
CD25À
T cells. Foxp3-
mRNA negatively correlated with (i) expression of HLA-DR
Table 3 Postonset day-wise functionality of Treg on Teff
cells in acute hepatitis E patients. AVH-E patients denoted
as A1–A5
Sample
ID
POD
days
% Teff cell
proliferation
%
Proliferation
(Treg+Teff)
%
Suppression
(Treg+Teff)
Treg:Teff cells ratio
1:1 1:4 1:1 1:4
A1 6 35.1 20.9 23.3 33.62 40.46
A2 8 37.3 18.1 19.2 48.53 51.47
A3 9 58.9 33.9 34.9 40.75 42.44
A4 11 41.5 14.5 25.2 39.28 65.06
A5 14 55.8 34.2 36.01 35.48 38.71

Fig. 3 Characterization of CD25+
Treg and CD25À
Teff on CD4+
T cells of patients and controls: Representative ﬂow
cytometry analysis of CD4+
CD25+
(Treg) and CD4+
25À
(Teff) gated lymphocytes is shown in (a) PBMCs from patients and
controls were gated on lymphocytes via their forward (FSC) and side scatter (SSC) properties, and CD4+
CD25+
(b)
CD4+
CD25À
(c) were gated to determine the third marker expression in the study population (d–q). Boxes represent
interquartile ranges, and vertical lines represent ranges and horizontal lines represent medians. n = numbers of patients, §
denotes AVH-E vs recovered, * denotes AVH-E vs control and # denotes recovered vs control. Data are represented as
mean Æ SD and P-values ≤0.05 calculated using Mann–Whitney U-test are considered signiﬁcant.

on CD4+
CD25À
T cells (r = À0.527, P = 0.036) and (ii)
expression of CD73 and CCR4 on CD4+
CD25+
T cells
(r = À0.501, P = 0.04 and r = À0.518, P = 0.04), respec-
tively (Fig. 4).
DISCUSSION
In the current study, we have assessed the functionality
of regulatory T cells and have also carried out direct
comparison of expression of conventional phenotype mark-
ers that are frequently associated with human Treg func-
tion, on Treg and Teff cells in hepatitis E for the first time.
The current study explores previous research on the
involvement of Treg cells in hepatitis E using a refined
approach to measure the surface/intracellular expression of
molecules involved in the Treg cell machinery by combin-
ing a stringent gating strategy for CD25+
and CD25À
cells
on CD4+
T cells.
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j) (k) (l)
Fig. 4 Correlation between Foxp3 expression at mRNA/protein, serum HEV IgM level and different subsets of Teff/Treg
cells in hepatitis E infection: In AVH-E, a negative correlation of IgM titre with CD39 expression on both Treg and Teff cells
(a, b). Foxp3 protein level positively correlated with TGF-b (c). In recovered individuals, CD154 negatively correlated
with HEV IgM levels (d). TGF-b levels positively correlated with expression of CD103 on Treg and Teff with CCR4 on Treg
(e, f, g). Foxp3 mRNA level positively correlated with CD69 on Treg (h) and CTLA4 on Teff cells (i). Foxp3 mRNA level
negatively correlated with CD73, CCR4 on Treg (j, k) and HLA on Teff cells (l). P-value was determined using Mann–
Whitney U-test, while r, the correlation coefficient, was calculated using Spearman’s correlation.

We had earlier shown the elevation of CD4+
CD25+
Foxp3+
and CD4+
CD25À
Foxp3+
in hepatitis E [4]. Recent studies
have focused on CD25 as the best marker for regulatory
CD4+
T cells in mice and humans [20]. It functions as acti-
vation-induced cytokine receptor and also has regulatory
function. A regulatory function has also been demonstrated
in the subsets negative for CD25 [21–23].
Our observation of increased CD3+
CD4+
T cells only in
AVH-E patients goes in parallel with the reports of NKT
and Treg mediated overall expansion of CD4+
T cells
[4,24]. Significant elevation of Foxp3 on CD8+
CD25+
and
CD8+
CD25À
T cells in the patient groups along with an
increase in CD3+
CD8+
T cells in the recovered group sug-
gests the involvement of CD8-mediated Treg cells and pro-
vides further scope for research of the same [16].
Significantly elevated frequencies of Treg and higher levels
of TGF-b in AVH-E and in recovered individuals led us to
assess the functions of Treg on Teff during the acute and
recovered phases of infection. Suppression of Teff cells was
more in the AVH-E patients than the recovered and con-
trols indicating that Treg cells in AVH-E might be having
the potential to suppress effector T-cell proliferation as seen
in other acute viral infections [25,26].
Significant elevation of Foxp3 at the mRNA and cellular
levels, high levels of IL-10, TGF-b in the patient groups go
in parallel with the positive correlation of TGF-b with the
expression of Foxp3.This indicated a possible role for TGF-b
in inducing Treg cells during HEV infection [27]. This sup-
ports the reports of association of the immunoregulatory
cytokines IL-10 and TGF-b with the generation of new
Treg cells [11] and the involvement of TGF-b in converting
peripheral CD4+
CD25À
na€ıve T cells to CD4+
CD25+
Treg
via induction of Foxp3 [28,29].
Next, we assessed the expression/involvement of certain
key conventional molecules on Treg and Teff cells. Sune-
etha et al. have recently shown impaired HEV-specific T-
cell responses in chronic hepatitis E and that the same
could be restored by blocking the PD1 or CTLA-4 path-
ways [30]. The current series of patients had elevated
expression of CTLA-4 and PD1 on both Treg and Teff cells
during the acute phase and that explains their adequate T-
cell response.
Higher expression of CD95 on Treg in patient groups war-
rants the need of further work to explore the CD95/CD95L
system for assessing its function as reported [31–33].
Increased GITR expression levels on both Treg and Teff cells
in the patient categories might be contributing to the expan-
sion of the same in peripheral circulation as reported [34].
Distinct expression of CD103 on Treg and Teff cells sug-
gests that CD103 could be a marker for regulatory CD4+
T-cell subpopulations in self-limiting hepatitis E as the
same has been shown to be an excellent marker for identi-
fying CD4+
regulatory T cells [35].
Deaglio et al. have identified CD39 together with CD73
as novel cell surface markers of CD4+
Treg cells and as an
important component of the Treg suppressive machinery
[14]. CD39 Treg has been positively correlated with HBV
load but inversely correlated with serum ALT level associ-
ating CD39 expression on Foxp3+
Treg cells with disease
progression in HBV infection [36]. CD39 and CD73 eleva-
tion on Treg cells of the patient groups, negative correla-
tion of CD39 expression on Treg and Teff cells of AVH-E
patients with IgM levels and no correlation of the same
with ALT levels and HEV RNA suggest that CD39+
Treg
populations are dynamic, but not associated with severity/
inflammation. Higher expression of CCR4, HLA-DR,
CD69, CD71, 4-1BB, CD70, CD40, CD154 and ICOS on
Treg/Teff cells need further experiments to assess individual
involvement.
Upregulation of Treg frequency is not associated with
increase in HEV replication or IgM and ALT levels in
hepatitis E patients [4]. The functional data obtained
from 5 acute patients suggested that there is no correla-
tion between the clinical natural history of the infection
and the function of Treg on Teff during acute HEV
infection.
One of the strengths of the current study is that the
CD4+
CD25+
CD127dim/À
Treg cells of hepatitis E patients
have the ability to suppress/inhibit autologous CD4+
CD25À
Teff cells. Certain peripheral Treg cell markers frequently
associated with human Treg function are also associated
with self-resolving hepatitis E infection. This study opens
avenues for the exploration of the Treg-mediated suppres-
sion mechanism and for assessing the functionality of con-
ventional markers on regulatory T cells, in acute viral
hepatitis E, in male and pregnant acute liver failure hepati-
tis E patients.
ACKNOWLEDGEMENTS
The authors thank to the Director, National Institute of
Virology for all the encouragements. Special thanks are
due to Mr. BN Tilekar, Mr. PB Jawalkar and Mr. PV Aya-
chit for technical help. Graphical assistance by Dr. MS
Paingankar is acknowledged. This study was funded by the
Indian Council of Medical Research, New Delhi, India
through a research grant to Dr. Anuradha S Tripathy.
Sanjay B Rathod was supported by a Junior Research Fel-
lowship (Inspire Fellow) grant from the Department of Sci-
ence and Technology, New Delhi, India.
FINANCIAL SUPPORT
This study was funded by the Indian Council of Medical
Research, New Delhi, India.
CONFLICTS OF INTEREST
The authors have declared that no competing interests
exist.

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