Anukumar and Shahir Virology Journal 2011, 8:259

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RESEARCH Open Access

Immune regulation in Chandipura virus infection:

characterization of CD4+ T regulatory cells from
infected mice
Balakrishnan Anukumar* and Prajakta Shahir

Abstract
Back ground: Chandipura virus produces acute infection in mice. During infection drastic reduction of CD4+, CD8
+ and CD19 + cell was noticed. Depletion of lymphocytes also noticed in spleen. The reduction may be due to
the regulatory mechanism of immune system to prevent the bystander host tissue injury. There are several
mechanisms like generation of regulatory cells, activation induced cell death (ACID) etc were indicated to control
the activation and maintain cellular homeostasis. Role of regulatory cells in homeostasis has been described in
several viral diseases. This study was undertaken to characterize CD4+T regulatory cells from the infected mice.
Method: In this study we purified the CD4+ T cells from Chandipura virus infected susceptible Balb/c mice. CD4+
T regulatory cells were identified by expression of cell surface markers CD25, CD127 and CTLA-4 and intracellular
markers Foxp3, IL-10 and TGF-beta. Antigen specificity and ability to suppress the proliferation of other
lymphocytes were studied in vitro by purified CD4+CD25+T regulatory cells from infected mice. The proliferation
was calculated by proliferation module of Flow Jo software. Expression of death receptors on regulatory cells were
studied by flowcytometer.
Results: The CD4+ T cells isolated from infected mice expressed characteristic markers of regulatory phenotype at
all post infective hours tested. The CD4+ T regulatory cells were proliferated when stimulated with Chandipura
virus antigen. The regulatory cells did not suppress the proliferation of splenocytes stimulated with anti CD3
antibody when co cultured with them. Interesting observation was, while purification of CD4+ T cells by negative
selection, the population of cells negative for CD4 also co purified along with CD4+ T cell. Flow cytometry analysis
and light microscopy revealed that CD4 negative cells were of different size and shape (atypical) compared to the
normal lymphocytes. Greater percentage of these atypical lymphocytes expressed Fas Ligand and Programmed
Death1 (PD-1) receptor.
Conclusion: From these results we concluded that virus specific CD4+T regulatory cells are generated during
Chandipura virus infection in mice and these cells might control the activated lymphocytes during infection by
different mechanism.

Background virus produces acute infection and induces reduction in

Chandipura virus belongs to the family Rhabdovirdae, the percentage of CD4, CD8 and CD19 positive cells in
genus vesiculovirus associated with acute encephalitis infected susceptible mice [6]. Lymphocytes reduction
and severe fatality in young children [1,2]. Children during acute viral infection is common in several viral
below 15 years old were vulnerable but adults were diseases [7-9]. After encountering the pathogen, the
refractory to the infection. The age dependent suscept- lymphocytes become activated and proliferate to elimi-
ibility has also been noticed in mice [3-5]. Chandipura nate the pathogen. This activated T lymphocytes are
continuously generated during immune response against
infection and it should be removed from the immune
* Correspondence: [email protected]
Chandipura Virus Group, National Institute of Virology, 20-A, Dr.Ambedkar
system to prevent the bystander tissue injury as well as
Road, Post Box No.11. Pune-411001, India to maintain the cellular homeostasis. There are several
© 2011 Anukumar and Shahir; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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mechanisms like generation of regulatory cells, activa- footpad. Uninfected healthy mice of same age group
tion induced cell death (ACID) etc were indicated to were kept as a control throughout the study. All the
control the activation and maintain cellular homeostasis. assays described in this study carried out till 72 h PI
Role of regulatory cells in homeostasis have been because most of the infected mice died at 96 h PI.
described in several reviews [10,11]. These cells serve to
limit the activation and effectors functions of CD4+ and Purification of splenocytes and staining for Flow
CD8 + T cells. These cells not only protect from auto- cytometry
immune disease but also protect from exogenous anti- Single cell suspension of splenocytes were prepared by
gen [12]. Several proposed mechanisms of regulatory teasing the spleen in nylon mesh containing 5 ml of
cells to limit T cell response include expression of IL- RPMI 1640 supplemented with 10% FCS, 25 mM
10, TGF-b, surface expression of CTLA-4 receptor, IL-2 HEPES and 5 × 10 -5 M b-mercaptoethanol (Growth
sequestration, blockade of co stimulatory molecules etc medium). The mononuclear cell population was purified
[13]. Regulatory T cells are diverse in nature and include by density gradient centrifugation using histopaque
at least three populations that differ by their phenotype, (1.083 gm/ml, Sigma) and washed twice in RPMI 1640
cytokine profile and suppressive mechanism [14]. medium. The cells were counted and stained with var-
The involvement of Fas-Fas ligand in AICD was well ious anti mouse antibody conjugated with different
reviewed by Stephen Maher et al [15]. As part of the fluorescent molecules. Acquisition and analysis were
host cell defense mechanism, it may reduce virus growth done in FACScalibur using cell quest pro soft ware (BD
as well as it spread and dissemination within the organ- Bioscience). In some analysis FlowJo software (Tristar)
ism. Recent studies have shown that the Programmed was used. The lymphocyte population was gated in for-
death 1 (PD-1) inhibitory pathway plays a critical role in ward scatter (FSC) vs side scatter (SSC) dot plot. The
modulating the functional exhaustion of virus specific T CD3 positive cells were further gated from the lympho-
cells and play a dual role in immune regulation by pre- cyte population in FSC vs CD3+ dot plot. CD3+CD4+
venting the attack on self and keeping activated immune population was analyzed for expression of different
system in check. This has now been documented in sev- markers.
eral animal models such as murine Lymphocytic Chor-
iomeningitis virus (LCMV) infection [16] and simian Purification of CD4+ cells from splenocytes
immunodeficiency virus (SIV) infection of non-human Splenocytes from infected as well as uninfected mice
primates [17] and, more importantly, in humans during were isolated as described above. The CD4+ cells were
persistent infection with human immunodeficiency virus purified from the splenocytes by using mouse CD4+ cell
(HIV), hepatitis B virus (HBV) and hepatitis C virus isolation kit (Miltenyi Biotec) as per the manufacturer’s
(HCV) [18,19]. instruction. The purity was checked by staining with
This study is focused on whether the CD4+ T regula- anti mouse CD4 antibody conjugated with fluorescein
tory cells induced during experimentally Chandipura isothiocyanate (FITC) and analyzed in FACScaliber.
virus infected mice or not. These cells play an important
role in cellular homeostasis. Chandipura virus specific Phenotypic characterization of CD4+ T cells from infected
CD4+ T regulatory cells induced during infection. mice
Surface receptor and ligand expression
Methods The purified CD4+ cells were stained with anti mouse
Cells, virus and animals CD4, CD25, CD127 and CTLA4 (CD152) antibody con-
The strain 034267 was originally isolated from encepha- jugated with different fluorescent molecules
litis outbreak in Andhra Pradesh, India, 2003. The virus (eBioscience). The stained cells were analyzed in FACS-
was propagated and titrated in Vero E6 cells and main- caliber. The percentage of CD4+ cells expressing differ-
tained in our laboratory. The Vero E6 cells were ent markers was calculated by following formula
obtained from National Center for Cell Science, India
and were grown in DMEM (Pan Biotech) supplemented % of marker positive CD4 + cells = No. of marker positive CD4 + cells in analyzed sample×
100/No. of total CD4 + cells in analyzed sample
with 10% Fetal Calf Serum (FCS, Hyclone). Balb/c mice
maintained in institute animal house were used in this
study. The mice at 13 days old were used in this experi-
ment. The mice were maintained in adlibidum water Quantitation of Foxp3, TGF-b, IL-10 transcripts
and feed throughout the experiment. The study was Total RNA was extracted from one million CD4+ T
approved by institute animal ethical committee (IAEC). cells using PureLink RNA mini kit (Invitrogen) accord-
For experimental infection, the mice were inoculated ing to the manufacturer’s instructions. The RNA was
with 10 μl (titer 5 log LD50/100 μl) of virus through eluted in 50 μl volume and 8 μl was used for cDNA
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synthesis. The level of expression of Foxp3, IL-10 and Statistical analysis

TGF-b was quantitated by SYBR green based real time Student’s t test was used to compare the treated and
PCR using published primers [13]. Two μl of cDNA was control groups. The p value less than 5% was considered
used for amplification using MESA green qPCR kit as significant.
(Eurogentech). To normalize the data, Cyclin A, the
housekeeping gene was amplified. Relative fold change Results
of expression of different transcript was calculated by Induction of CD4+T regulatory cells during infection
formula 2- ΔΔCt . The ΔΔCt was calculated from unin- The CD4+ T regulatory cells can be differentiated from
fected control. CD4+T cells by level of expression of CD25 and CD127
on cell surface. Moreover these cells also expresses the
Expression of death receptors transcription factor Foxp3 and the cytokines IL-10 and
The purified cells were also stained with anti mouse Fas TGF-b. These cytokines play a major role in regulation
(CD95), Fas ligand (CD95L), PD-1, TRANCE, and of immune system. In Chandipura infected mice CD4
CD40L antibody conjugates. The stained cells were ana- +CD25+ T cells were noticed from 24 h PI onwards and
lyzed by flow cytometer as mentioned above. The per- the level was significantly high at 72 h PI (4%, p < 0.01)
centage of CD4+ or CD4- cells positive for above (Figure 1). The CD4+CD127+T cell level was decreased
mentioned receptors were calculated. from 24 h PI onwards and it was lower than control at
72 h PI (Figure 2). No significant difference was noticed
Functional characterization of CD4+T regulatory cells in CTLA-4 expression (Figure 3). The level of expres-
Purification of CD4+CD25+ regulatory T cells (T regs) sion of Foxp3, IL-10 and TGF-b was quantitated by
Splenocytes from infected as well as uninfected animal SYBR green based real time RT-PCR. The result indi-
were purified as described above. The CD4+CD25+ T cated that the expression of these transcripts were
cells were purified from the splenocytes by using mouse noticed in all PI hours tested (Figure 4).
CD4+CD25+ T regulatory cell isolation kit (Miltenyi
Biotec) as per the manufacturer’s instruction. Antigen specificity of T regs
T reg proliferation assay T regs cells isolated from both control and infected
The purified T regs were labeled with carboxyfluores- mice were stimulated with inactivated whole Chandi-
cein succinimidyl ester (CFSE) as per the standard pro- pura virus antigen. Proliferation was calculated by
cedure. Briefly, 20 million cells in one ml of PBS+0.1% Flow Jo software. It is evident that proliferation of
BSA was stained with 5 μM final concentration of CFSE CD4+CD25+T cells in vitro because their level of the
and incubated 15 min in RT. The reaction was stopped CFSE dye, which is divided equally among daughter
by addition of equal volume of FCS. The cells were cells upon cell division, has decreased. The percentage
washed twice with growth medium. The final cell pellet of CD4+CD25+T cells in different daughter population
was seeded in to the 24 well plate containing inactivated was calculated. The percentage of CD4+CD25+ T cells
Chandipura virus whole antigen pulsed splenic macro- from infected mice was 7.72% in first generation
phages. T regs from control mice was also treated in the daughter population and it was 3.39% in control mice
same way. The cells were incubated for three days at 37° (p < 0.001). Similarly in second generation daughter
C in 5% CO2. After incubation, the cells were stained cell it was 2.18% in infected and 1.24% in control (Fig-
with anti mouse CD4-PE-Cy7 and acquired in FACScali- ure 5).
ber and analyzed in Flow Jo software using proliferation
module. Suppression of proliferation of CD4+T cells by T regs
No significant suppression was noticed in anti CD3 anti-
Suppression assay body stimulated splenocytes from normal mice co cul-
Splenocytes from normal mice were labeled with CFSE tured with T regs from infected mice (Figure 6).
as described above. The labeled splenocytes were co cul-
tured with T regs from infected mice in the presence of CD4 negative population in infected mice
1 μg/ml of mouse anti CD3 antibody (eBioscience). While purification of CD4+T cells by negative selec-
Appropriate control including only splenocytes and sple- tion, cells negative for CD8, CD11b, CD45R, CD49b
nocytes with anti CD3 antibody also kept in the plate. and Ter-119 also purified along with CD4+Tcells. The
The cells were incubated for five days at 37°C in 5% CD4- population was approximately 10% more in
CO2. After incubation the cells were stained with anti infected mice (Figure 7). Leishman’s staining of this
mouse CD4-PE-Cy7 conjugate and acquired in flow cyt- cell showed different morphology with distributed
ometer. The acquired data was analyzed by flow Jo soft chromatin (Figure 8). Both the CD4+ and CD4- cells
ware using proliferation module. were stained with Fas, Fas ligand, PD-1, CD40L,
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Figure 1 Expression of CD25 on CD4+T cells from mice. The CD4+ T cells from infected and control mice were purified at different hours
post infection (PI) and stained with anti mouse CD25 antibody conjugated with phycoerythrin (PE). The percentage of CD4+ T cells positive for
CD25 were calculated by formula mentioned in methods. The biexponential transformation of dot plot represents the percentage of CD4+
population which showed significant changes in expression of CD25 receptor at different hour post infection. Values in upper right corner are
Mean ± SE of three independent experiments,*p < 0.01
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Figure 2 Expression of CD127 on CD4+T cells from mice. The CD4+ T cells from infected and control mice were purified at different hours
post infection (PI) and stained with anti mouse 127 antibody conjugated with phycoerythrin-Cy7 (PE-Cy7). The percentage of CD4+ T cells
positive for CD127 were calculated by formula mentioned in methods. The biexponential transformation of dot plot represents the percentage
of CD4+ population which showed expression of CD127 receptor at different hour post infection. Values in upper right corner are Mean ± SE of
three independent experiments.
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Figure 3 Expression of CTLA-4 on CD4+T cells from mice. The CD4+ T cells from infected and control mice were purified at different hours
post infection (PI) and stained with anti mouse CTLA-4 antibody conjugated with allo-phycocyanin (APC). The percentage of CD4+ T cells
positive for CTLA-4 were calculated by formula mentioned in methods. The biexponential transformation of dot plot represents the percentage
of CD4+ population which showed expression of CTLA-4 receptor at different hour post infection. Values in upper right corner are Mean ± SE of
three independent experiments.
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14 35
30
12

% of CD4+T cells
25
10
relative fold change

20
8 15
Foxp3
6 10
IL-10
5
TGF-beta
4
0
2 0th 1st 2nd
Daughter population
0
24 48 72 splenocytes splenocytes + CD3 splenocytes + T reg + CD3
HPI Figure 6 Proliferation of anti CD3 antibody stimulated
Figure 4 Expression of Foxp3, IL-10 and TGF-b transcripts in splenocytes in the presence of CD4+CD25+ T cells (T regs)
CD4+T cells from infected mice. The CD4+ T cells from infected from infected mice. The splenocytes were stained with CFSE and
and control mice were purified at different hours post infection the proliferation was calculated on 5th day post stimulation by
(HPI) and the expression of Foxp3, IL-10 and TGF-b transcripts were proliferation module. The values are Mean ± SE of triplicate.
quantitated by SYBR green based real time RT-PCR. The relative fold
change (two fold) was calculated from uninfected control as
mentioned in methods. The values are Mean ± SE of three receptor and in control it was 12% (p < 0.01). The
independent experiments. PD-1 receptor expression was noticed in entire PI
hours tested (Figure 10). No significant changes
TRANCE and CD69 markers. Approximately 25% of between infected and control was noticed in other
CD4- cells expressed Fas ligand compared to CD4- receptors tested in this experiment. Similarly no sig-
cells from control (8%) at 24 h PI (p < 0.05) (Figure nificant changes in expression were noticed in these
9). The difference in expression was also noticed at 48 receptors in CD4+ cells also (data not shown).
h PI. Similarly 38% of this cell also expressed PD-1
Discussion
Silencing of T cell response to acute viral infection is
16 essential to maintain the homeostasis of immune cells
14
and also to avoid the untoward effects on bystander
cells. Because of this reason, several acute viral infec-
% of CD4+CD25+ T cells

12
tions produces lymphocyte reduction in host [7-9]. In
10 p<0.01 Influenza A infection in human it was reported that
8 virus replication in lymphocytes leads to reduction of
Treg(con)+ antigen
6 Treg(inf)+ antigen
lymphocytes [7]. In this study the mechanism behind
4
the reduction was analyzed with focus on T regulatory
cells.
2
The CD4+T cells from infected mice expressed CD25,
0
Foxp3+, IL-10 and TGF-b, the characteristic markers of
0st 1st 2nd
Daughter Population
regulatory phenotype of T cells. This regulatory popula-
tion of cells noticed from 24 h PI onwards and highest
Figure 5 In vitro Chandipura antigen specific proliferation of
CD4+CD25+ T cells (T regs) from infected and control mice. number of population noticed at 48 and 72 h PI. Chan-
The double positive cells were purified and stained with CFSE and dipura viral antigen specific proliferation of these cells
co cultured with splenic macrophages pulsed with Chandipura indicated that these cells are virus specific. These cells
antigen. The proliferation was calculated at 72 h post infection by were activated during infection but did not suppress the
proliferation module in Flow Jo software. It is evident that
proliferation of CD3 stimulated normal splenocytes in
proliferation in vitro because their level of the CFSE dye, which is
divided equally among daughter, cells upon cell division has vitro.
decreased. The percentage of CD4+CD25+T cells in different Interesting observation during purification of CD4+
daughter population was calculated. The values are Mean ± SE of cell was that the cells negative for CD4 and all other
triplicate. markers used in depletion was co purified along with
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Infected Control

CD4+T CD4+T
CD4-T CD4-T

CD4-FITC
Figure 7 Percentage of CD4 negative population of cells which was co purified along with CD4+ cells purified by negative selection
from infected and control mice. In histogram CD4-T marker represents the percentage of cells unstained with mouse anti CD4 antibody and
CD4+T marker represents percentage of cells stained with mouse anti CD4 antibody.

CD4+ cells. Morphologically these cells were larger than tor and Fas ligand. Fas L is a death ligand induces apop-
lymphocytes and showed various size and shape (atypi- tosis in cell that express Fas receptor. Similarly PD-1 is
cal). The chromatin was dispersed throughout the cells. extended family of CD28/CTLA-4 present on T cell reg-
These cells are called as atypical or reactive lymphocytes ulators [21,22] which negatively regulates the TCR sig-
and described by Simon (2003) [20] in his review. The nals. It is now known that up regulation PD-1 in
greater percentage of these cells expressed PD-1 recep- exhausted CD8 T cells is coincident with the progres-
sion of many chronic human diseases including human
immunodeficiency virus (HIV), hepatitis C virus, (HCV),
and Epstein Barr virus (EBV) [17,18,23]. Greater percen-
tage of PD-1 expression in acute Chandipura virus
infection indicated that immune system might suppress
the activated lymphocytes through PD-1 and PD-1
B ligand interaction to prevent bystander tissue injury.
The suppression of T cells might allow the infection to
progress because most of the infected mice died at 96 h
PI.
A Conclusion
This study concluded that different regulatory mechan-
isms activated during Chandipura virus infection in
mice. The induction of CD4+T regulatory cells and
expression of PD-1 may be one of the mechanisms by
x1000 which mice immune system control the activated lym-
Figure 8 Leishman’s staining of purified CD4+ T cells from phocytes and maintain the homeostasis. The exact role
infected mice. A. normal lymphocytes B. large size lymphocytes of these cells in immune regulation needs to be
with dispersed chromatin. studied.
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Figure 9 Expression of Fas ligand on CD4 negative T cell from mice. The CD4+ T cells from infected and control mice were purified at
different hours post infection (PI). The cells were stained with anti mouse Fas ligand antibody conjugated with allo-phycocyanin (APC). The cells
negative for CD4 receptors were gated and the percentage of CD4 negative T cells expressed the Fas ligand were calculated by formula
mentioned in methods. The biexponential transformation of dot plot represents the percentage of CD4- population which showed significant
changes in expression of Fas ligand at different hour post infection (PI). a p < 0.05
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Figure 10 Expression of Programmed Death 1 (PD-1) on CD4 negative T cell from mice. The CD4+ T cells from infected and control mice
were purified at different hours post infection (PI). The cells were stained with anti mouse PD-1 antibody conjugated with phycoerythrin-Cy7
(PE-Cy7). The cells negative for CD4 receptors were gated and the percentage of CD4 negative T cells expressed the PD-1 were calculated by
formula mentioned in methods. The biexponential transformation of dot plot represents the percentage of CD4- population which showed
significant changes in expression of PD-1 at different hour post infection (PI).b p < 0.01
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