IMMUNOLOGY
ORIGINAL ARTICLE
Protection conferred by heterologous vaccination against
tuberculosis is dependent on the ratio of CD4+/CD4+ Foxp3+ cells
Paola Fernanda Fedatto,1 Cássia
Alves Sérgio,1 Marina Oliveira e
Paula,1 Ana Flávia Gembre,1 Luı́s
Henrique Franco,1 Pryscilla Fanini
Wowk,1 Simone Gusmão Ramos,2
Cynthia Horn,3 Gilles Marchal,4
Walter Miguel Turato,1 Célio Lopes
Silva,1 Denise Morais da Fonseca1
and Vânia Luiza Deperon Bonato1*
1
Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto,
University of São Paulo, Ribeirão Preto, ,
2
Department of Pathology, School of Medicine
of Ribeirão Preto, University of São Paulo,
Ribeirão Preto, , 3Evandro Chagas Research
Institute, Osvaldo Cruz Foundation, Rio de
Janeiro, Brazil and 4Immunotherapix Bio
Top, Institute Pasteur, Paris, France
doi:10.1111/imm.12006
Received 09 April 2012; revised 08 June
2012; accepted 24 July 2012.
*Correspondence: Professor V. L. D. Bonato,
Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto,
University of São Paulo, Av. Bandeirantes,
3900, Ribeirão Preto, São Paulo 14049-900,
Brazil. Email: vlbonato@fmrp.usp.br
Senior author: Vânia Luiza Deperon Bonato
Summary
CD4+ Foxp3+ regulatory T cells inhibit the production of interferon-c, which
is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three
different vaccines against tuberculosis was associated with the number of
spleen and lung regulatory T cells. We observed that after homologous
immunization with the 65 000 molecular weight heat-shock protein (hsp 65)
DNA vaccine, there was a significantly higher number of spleen
CD4+ Foxp3+ cells compared with non-immunized mice. Heterologous
immunization using bacillus Calmette–Guérin (BCG) to prime and DNAhsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate
proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher
ratio of spleen CD4+/CD4+ Foxp3+ cells compared with non-immunized
mice. In addition, the protection conferred by either the BCG/DNA-hsp 65
or the BCG/CFP-CpG vaccines was significant compared with the DNAhsp 65 vaccine. Despite the higher ratio of spleen CD4+/CD4+ Foxp3+ cells
found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice,
the lungs of both groups of mice were better preserved than those of DNAhsp 65-immunized mice. These results confirm the protective efficacy of
BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines
and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost
regimens assayed here represent a promising strategy for the development of
new vaccines to protect against tuberculosis because they probably induce a
proper ratio of CD4+ and regulatory (CD4+ Foxp3+) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.
Keywords: CD4+ Foxp3+; DNA vaccine; heterologous immunization; regulatory T cell; tuberculosis.
Introduction
Effector CD4+ lymphocytes are divided into at least three
different subpopulations: T helper type 1 (Th1), Th2 and
Th17 cells. The functions of each of these groupings are
highly specialized and are controlled by regulatory CD4+
T cells. Regulatory T cells are further classified into three
different populations that inhibit either the innate or the
adaptive immune response.1 Regulatory T1 cells secrete
interleukin-10 (IL-10), Th3 cells secrete transforming
growth factor-b (TGF-b) and CD4+ Foxp3+ T cells may
play different roles.2–4 CD4+ Foxp3+ cells secrete IL-10
and/or TGF-b and suppress the activation of T lymphocytes, macrophages and dendritic cells.5–7 They also
express high levels of cytotoxic T-lymphocyte antigen-4.
This protein’s interaction with the B7 molecule triggers
the inhibition of antigen-presenting cell function.5,7,8
High levels of CD25, the a-chain of the high-affinity
receptor for IL-2 that is expressed on the membrane surface of CD4+ Foxp3+ cells promotes the sequestration of
IL-2 and inhibits the proliferation of T cells.9 There are
many other mechanisms that CD4+ Foxp3+ cells use to
inhibit T cells and antigen-presenting cells, such as cytotoxicity, the induction of the indoleamine 2,3-dioxygenase
enzyme and CD39 and CD73 expression.8,10
Regulatory T cells were first described in autoimmune
processes where the absence of CD25+ cells was associated
with the development of autoimmune disease.11–14 The
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
239
P. F. Fedatto et al.
description of the Foxp3 transcription factor indicated that
it was a key protein involved in the development, differentiation and characterization of regulatory T cells.15–18 Besides
autoimmune diseases, CD4+ Foxp3+ cells were found to
play a role in various other infectious diseases, such as tuberculosis. An increase in the number of circulating regulatory
T cells in tuberculosis patients was first described in
2006.19,20 The increase in the frequency of these cells was
directly associated with the inhibition of interferon-c
(IFN-c) secretion. The treatment of patients infected with
Mycobacterium tuberculosis resulted in a decrease in the
number of regulatory T cells and restored the production of
IFN-c.20,21 In experimental tuberculosis model, the depletion of Foxp3+ cells in infected C57BL/6 mice resulted in
fewer bacteria (measured as colony-forming units) in the
lungs compared with mice with Foxp3+ cells.22 While
C57BL/6 mice exhibited an increased magnitude of their
Th1 response and a lower effector function of their regulatory T cells, BALB/c mice had a lower magnitude of Th1
response and effector function of their regulatory T cells that
suppressed IL-2 and IFN-c secretion. This finding suggested
that regulatory T cells may potentially represent a susceptibility factor in tuberculosis.23 Recently, it was reported that
regulatory T cells delayed the migration of effector CD4+
and CD8+ cells to the lungs of infected mice.24
Tuberculosis remains a priority for public health. Each
year, 11 million individuals die from this disease.25
Intense efforts have been concentrated in the development
of new vaccines against tuberculosis. During decades of
research, an effective immune response against tuberculosis should be characterized by the induction of IFN-c-producing Th1 cells. Recently, the immunogenicity of vaccines
has also begun to be analysed to better characterize the
participation of Th17 and regulatory T cells.26,27
For years, we have worked with an experimental model
of M. tuberculosis infection in an attempt to study vaccines against tuberculosis. We have used protein and
microbial adjuvant, DNA vaccine and prime-boost heterologous immunization to improve vaccine-mediated
protection against M. tuberculosis infection.
In this study, we addressed the question of whether different vaccines designed to induce protection against
experimental tuberculosis would be able to induce
CD4+ Foxp3+ regulatory T cells. In addition, we compared the protective efficacy with the frequency of
regulatory T cells in the lungs of immunized and
challenged mice.
Materials and methods
Mice
Specific-pathogen-free, female BALB/c mice were bred in
the Animal Facility of the School of Medicine of Ribeirão
Preto, University of São Paulo, maintained under barrier
240
conditions in a Level III biohazard laboratory and used at
6–8 weeks of age.
The use of animals in this study was approved by the
Ethics Committee on Animal Experimentation, protocol
no. 056/2007.
M. tuberculosis antigens
Mycobacterium tuberculosis culture filtrate proteins (CFP)
were provided by Gilles Marchal, Pasteur Institute, Paris,
France and sonicated M. tuberculosis (Mtb antigen) was
obtained from H37Rv strain (ATCC 27294; American
Type Culture Collection, Rockville, MD). Briefly, mycobacteria were cultured as previously described,28 heatkilled, sonicated for 15 min and centrifuged at 5000 g for
30 min. The supernatant was sterilized by filtration and
the protein concentration was quantified with the Coomassie Plus assay kit (Pierce, Rockford, IL).
CpG oligodeoxynucleotides
The CpG oligodeoxynucleotides 1826 were synthesized by
Invitrogen (Invitrogen Corp., Custom Primers, Carlsbad,
CA) at a 1-lmol scale with a phosphorothioate link in all
bases except the last one, according to the following
sequence: 5′-TCC ATG ACG TTC CTG ACG TT-3′.
Plasmid construction and immunizations
The DNA vaccine pVAX-hsp 65 (i.e. 65 000 molecular
weight heat-shock protein) (DNA-hsp 65) was derived
from the pVAX vector (Invitrogen) and was constructed
as described previously.28 For the DNA vaccination,
50 lg DNA-hsp 65 in 50 ll saline solution plus 125%
sucrose was injected into each quadriceps muscle (a total
of 100 lg per dose) for three doses at 15-day intervals.
For the heterologous, prime-boost immunization regimen, a single dose of bacillus Calmette–Guérin [BCG;
2 9 105 colony-forming units (CFU)] in 100 ll pyrogenfree saline was administered by the subcutaneous (s.c.)
route and after 15 days a single dose of the DNA-hsp 65
vaccine (100 lg) was administered by the intramuscular
(i.m.) route. In the second strategy of prime-boost heterologous immunization, the mice received the first immunization with subcutaneous (s.c.) BCG, as described
previously, and after 15 days received a single dose of s.c.
CFP (50 lg) and CpG (50 lg) at a final volume of
100 ll. The dose of 50 lg CFP and 50 lg CpG was standardized previously.29 Control groups were as follows: a
group that received an equal volume of saline; a group
immunized with a single dose of s.c. BCG (2 9 105 CFU)
and a group that received the pVAX plasmid without the
hsp 65 gene.
To evaluate the number of CD4+ Foxp3+ cells in the
spleens of different groups of immunized mice, recombinant
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
Heterologous vaccination against tuberculosis and CD4+Foxp3+ cells
hsp 65 protein was administered by the intravenous route
15 days after the completion of the immunization schedule
with either the DNA-hsp 65 vaccine or the BCG/DNAhsp 65 vaccine.
Bacteria and infection
The M. tuberculosis suspension was prepared and inoculated by the intratracheal route (1 9 105/mouse) as previously described.30
CFU determination
Thirty days after infection, the lower and medium right
lobes of the lungs were cut into small pieces and digested
with Liberase Blendzyme 2 (05 lg/ml; Roche, Indianapolis, IN) and deoxyribonuclease I (25 U/ml; GIBCO BRL,
Grand Island, NY) diluted in incomplete RPMI-1640.
Samples were incubated at 37° for 30 min and serial dilutions of the digested lobes were plated onto supplemented
7H11 agar media (BD Bioscience, Franklin Lakes, NJ).23
The number of CFU was determined 30 days after incubation at 37°.
Flow cytometry
The lung cell suspension was passed through a filter to
remove pieces of non-digested lung, centrifuged and
resuspended in complete RPMI-1640. Spleens were
homogenized, and the spleen cell suspensions were resuspended in RPMI-1640 (Sigma-Aldrich, St Louis, MO).
Lung (5 9 105 to 1 9 107) and spleen cells (1 9 106)
were incubated for 40 min at 4° with Fc Block (1 lg/
1 9 106 cells; BD Bioscience, Franklin Lakes, NJ). Cells
were then incubated with anti-CD4 (clone RM4-5) for
30 min at 4° in total darkness. After staining for surface
protein, the staining of Foxp3 was performed using the
FITC anti-mouse/rat Foxp3 Staining kit (eBioscience,
San Diego, CA) according to the manufacturer’s
instructions. The cells were analysed using flow cytometry with the FACSCantoTM (Becton Dickinson and
Company-Immunocytometry Systems, San Jose, CA).
The frequency of each cell population (CD4+ and
CD4+ Foxp3+) was calculated in lymphocytes gated by
size and granularity. The absolute number of the cell
population (CD4+ or CD4+ Foxp3+) was calculated by
the percentage of total events acquired in the samples
and the total number of cells obtained from the
Neubauer chamber.
Histology
For histopathological analysis, the right upper lobes of
the lungs were fixed in 10% formalin, embedded in paraffin blocks, prepared routinely and then sectioned for light
microscopy. Five-micrometre sections were stained with
haematoxylin & eosin.
Delayed-type hypersensitivity (DTH)
Regulatory T cells (CD4+ CD25+) were purified from the
spleen of non-immunized, immunized (15 days after
immunization), infected (30 days after infection) or uninfected mice with the CD4+ CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany) according to the manufacturer’s instructions.
The cells were resuspended (5 9 105 to 6 9 105 cells)
with 50 lg sonicated M. tuberculosis (Mtb Ag) in 40 ll
endotoxin-free sterile saline and injected in the right paw.
As a control, mice were injected with 40 ll saline in the
left paw or mice were injected only with Mtb antigen. The
footpads were measured with a precision micrometer at 0,
24, 48 and 72 hr after the administration of antigen and
CD4+ CD25+ cells. The ∆ DTH was quantified by measuring the difference between the footpad thickness over time.
Anti-CD25 treatment
Mice were injected intraperitoneally with 500 µg rat IgG1
anti-mouse CD25 monoclonal antibody (mAb) (PC61) or
rat immunoglobulin G (immunoglobulin control), kindly
donated by Dr João Santana da Silva (FMRP-USP). Either
the PC61 mAb or the immunoglobulin control was
administered 3 days before each immunization and
3 days before infection. Depletion was assessed using flow
cytometry of peripheral blood samples after the second
administration of the PC61 mAb.
Statistical analysis
All values are expressed as the mean ± standard error of
the mean (SEM). The PRISMA software (Graph Pad software, Inc., San Diego, CA) was employed for all analyses.
We used the Kolmogorov–Smirnov test and verified that
the values were in a Gaussian distribution. Next we used
one-way analysis of variance and Newman–Keuls multiple
comparison test. A value of P < 005 was considered significant. The correlation between the ratio of CD4+/
CD4+ Foxp3+ cells and CFU counts, and the number of
CD4+ Foxp3+ cells and CFU counts were determined by
the Pearson′s correlation coefficient.
Results
DNA-hsp 65 immunization, but not prime-boost
strategies, induced the expansion of spleen
CD4+ Foxp3+ cells
The experimental protocol in Fig. 1(a) describes the
following three vaccines used: DNA-hsp 65, BCG/
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
241
P. F. Fedatto et al.
(a) Experimental protocol
Day 30
Day 0
Day 15
DNA-hsp 65 i.m.
pVAX i.m.
DNA-hsp 65 i.m.
BCG s.c.
BCG s.c.
pVAX i.m.
Saline
BCG s.c.
Saline
Day 42
Day 45
DNA-hsp 65 i.m.
rHSP65 i.v.
DNA-hsp 65 i.m.
CFP/CpG s.c.
pVAX i.m.
CD4+ and CD4+Foxp3+
Controls
analysis
Saline
Saline
CD4+
CD4+Foxp3+
(b)
60
*
Total number of spleen
CD4+ cells (×106)
50
14
*
*
&
12
40
*
10
8
30
6
20
4
10
2
0
0
Total number
Frequency
(c)
7
6
&
&
6
4
3
3
2
2
1
1
0
0
FP
BC
G
/C
BC
hs
AN
/D
G
BC
pV
-C
65
p
65
p
hs
AN
D
G
4
AX
5
pG
5
Ratio CD4+/CD4+Foxp3+
spleen cells (%)
Ratio CD4+/CD4+Foxp3+
spleen cells (Total number)
7
DNA-hsp 65 and BCG/CFP-CpG. Subcutaneous BCG was
used as the gold standard. In addition to BCG, we used
pVAX as an experimental control group. First, we evaluated the number of spleen CD4+ cells after immunization.
DNA-hsp 65, BCG/DNA-hsp 65 and BCG/CFP-CpG vaccinations induced an increase in the number of CD4+
cells compared with non-immunized mice (black dotted
line). In addition, the higher number of CD4+ cells
induced by DNA-hsp 65 immunization was significant
compared with the BCG-immunized group (Fig. 1b).
There was also no difference in the frequency of spleen
CD4+ cells (data not shown). Immunization with the
242
16
*
Total number of spleen
CD4+Foxp3+ cells (×106)
Figure 1. Total number and frequency of
spleen CD4+ and CD4+ Foxp3+ cells from
immunized mice. BALB/c mice were immunized with the DNA-heat-shock protein 65
(hsp 65), BCG/DNA-hsp 65 and BCG/CFPCpG vaccines. Mice were immunized with
BCG or injected with pVAX plasmid or saline
as experimental controls. Fifteen days after the
completion of the immunization schedule, the
total number and frequency of effector (CD4+)
and regulatory (CD4+Foxp3+) T cells were
evaluated in the spleen (a). Spleen cells were
first gated by size (forward scatter) and granularity (side scatter) and then by CD4 and
Foxp3 expression. The results are expressed as
the means ± SEM of individual animals analysed in each different group. Total number of
spleen CD4+ cells and total number of spleen
CD4+ Foxp3+ cells (b). Ratio of total CD4+/
CD4+ Foxp3+ cells and ratio of the frequency
of CD4+/CD4+ Foxp3+ cells (c). The total
number of cells is representative of two independent experiments with the following experimental n: DNA-hsp 65, 6; BCG/DNA-hsp 65,
8; BCG/CFP-CpG, 6; pVAX, 7; BCG, 4. The
cell frequency data are representative of three
independent experiments (DNA-hsp 65, 8;
BCG/DNA-hsp 65, 8; BCG/CFP-CpG, 6;
pVAX, 10; BCG: 12). *P < 005 compared with
non-immunized mice (black dotted line).
&P < 005 compared with non-immunized
mice (grey dotted line). Horizontal bars represent the significant difference between groups.
Experimental
control groups
DNA-hsp 65 vaccine stimulated an increase in the number of spleen CD4+ Foxp3+ cells compared with the nonimmunized (grey dotted line), BCG prime DNA-hsp 65
boost and BCG groups (Fig. 1b). The frequency of these
cells was similar among the different experimental groups
(data not shown). We determined the ratio of CD4+ to
CD4+ Foxp3+ cells and found that the heterologous
prime-boost vaccination strategies (BCG/DNA-hsp 65
and BCG/CFP-CpG) elicited a significant increase in the
ratio of CD4+/CD4+ Foxp3+ cells, but these regimens did
not increase the total cell number (Fig. 1c) compared
with other groups. Therefore, DNA- hsp 65 was the
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
Heterologous vaccination against tuberculosis and CD4+Foxp3+ cells
vaccine that predominantly induced a higher number of
CD4+ Foxp3+ regulatory T cells, whereas the three vaccines DNA-hsp 65, BCG/DNA-hsp 65 and BCG/CFPCpG induced a significant increase of spleen CD4+ cells.
Indeed, the change in the ratio of CD4+/CD4+ Foxp3+
cells revealed that the heterologous prime-boost immunization was more effective in inducing a frequency of
CD4+ cells significantly higher than the frequency of
CD4+ Foxp3+ cells compared with non-immunized mice
and the BCG-immunized group. Spleen cell cultures
showed that the immunization with DNA-hsp 65, BCG/
DNA-hsp 65 and BCG/CFP-CpG preferentially induced
antigen-specific IFN-c secretion instead of IL-10 (data not
shown).
Lower numbers of lung CD4+ Foxp3+ cells were
correlated with a stronger protection conferred by
vaccination
Different groups of mice were immunized and challenged
with M. tuberculosis according to the experimental protocol (Fig. 2a). In the lungs, all different vaccines studied
were protective compared with the non-immunized,
infected group (black dotted line; Fig. 2b). However, the
BCG/DNA-hsp 65 and BCG/CFP-CpG immunizations
induced a significant decrease in lung CFU counts compared with the DNA-hsp 65 vaccine. A similar reduction
in CFU counts was also observed in the spleens of
immunized and challenged mice compared with the nonimmunized, infected mice group (grey dotted line;
Fig. 2b). Next, we quantified the number of CD4+ and
CD4+ Foxp3+ regulatory T cells in the lungs of immunized and challenged mice and verified that there was no
significant difference in the number of both cell populations (Fig. 2c,d). Despite the lower number of
CD4+ Foxp3+ cells in the lungs of BCG/DNA-hsp
65- and BCG/CFP-CpG-immunized mice, this was not
significantly different from non-immunized, infected
mice, DNA-hsp 65 immunized mice or BCG-immunized
mice (Fig. 2d). We also determined the ratio of lung
CD4+/CD4+ Foxp3+ cells and verified that no significant
difference existed among the different groups, despite the
higher ratio CD4+/CD4+ Foxp3+ cells observed in the
lungs of BCG/DNA-hsp 65- and BCG/CFP-CpG-immunized groups. These results show a tendency of the primeboost immunization strategies to expand and recruit more
CD4+ cells than CD4+ Foxp3+ cells to the lungs of challenged mice (Fig. 2e). We also established correlations
between the ratio of total number of lung CD4+/
CD4+ Foxp3+ cells and CFU counts, and between the
total number of lung CD4+ Foxp3+ cells and CFU counts.
We observed a moderate negative correlation (Pearson′s
correlation, r = 0356, P = 0212) between the ratio of
CD4+/CD4+ Foxp3+ cells and CFU counts (Fig. 2f) and a
moderate positive correlation (Pearson′s correlation,
r = 0404, P = 0152) between CD4+ Foxp3+ cells and
CFU counts (Fig. 2g). These data suggest that a lower
number of lung CD4+ Foxp3+ cells is associated with the
protection conferred by vaccines against tuberculosis.
BCG/DNA-hsp 65-immunized mice had better
preserved lungs than DNA-hsp 65-, BCG/CFP-CpGand BCG-immunized mice
We also evaluated the preservation of lung parenchyma
in non-immunized, infected mice and in the different
groups of immunized, challenged mice (Fig. 3). Experimental controls indicated that after 30 days of infection
there was a multifocal and diffuse inflammation in the
lungs of non-immunized, infected mice. The lungs of
infected mice were characterized by the presence of foamy
macrophages, few neutrophils and hyperplasia of the
bronchus-associated lymphoid tissue (BALT), which
indicated a higher number of lymphocytes compared with
the uninfected group. The lungs of the DNA-hsp 65vaccinated mice (DNA-hsp 65) were similar to the
infected group and had a higher number of neutrophils
compared with the lungs of infected mice. The lungs of
the control immunization group, which received pVAX
vector (pVAX) injection, were characterized by higher
inflammation compared with the DNA-hsp 65-immunized mice. The lungs of the BCG/CFP-CpG-immunized
mice (BCG/CFP-CpG) exhibited a discrete inflammatory
infiltrate, reduction in BALT and a decrease in the number of foamy macrophages compared with the lungs of
the infected group and the DNA-hsp 65-immunized mice.
BCG-immunized mice were similar to the BCG/CFPCpG-immunized mice. Mice immunized with BCG prime
and DNA-hsp 65 boost (BCG/DNA-hsp 65) had the
greatest preservation of the lungs: decreased BALT, a
reduction in foamy macrophages and neutrophils and
lower cellular influx compared with the infected group
and the BCG-immunized group. These results suggest
that while the BCG/DNA-hsp 65 vaccine induced a high
ratio of spleen CD4+/CD4+ Foxp3+ cells, this ratio seems
to be a key determinant in preventing disease progression
and reducing tissue damage.
Protection induced by the BCG/DNA-hsp 65 vaccine
was not affected by the depletion of CD25+ cells
To evaluate whether the number of CD4+ Foxp3+ cells
that were induced by vaccination affected both the function of these cells in vivo and their protective efficacy, we
performed a DTH assay to measure the activity of these
cells. A group of mice infected with M. tuberculosis
received Mtb antigen in the footpad 30 days after infection in the presence or absence of purified CD4+ CD25+
regulatory T cells isolated from uninfected (Saline Treg)
or infected (Mtb Treg) mice (Fig. 4a). Forty-eight hours
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
243
P. F. Fedatto et al.
Day 0
Day 30
Day 15
DNA-hsp 65 i.m.
30 days after
immunization
Day 60
pVAX i.m.
DNA-hsp 65 i.m.
BCG s.c.
BCG s.c.
pVAX i.m.
Saline
BCG s.c.
Saline
30 days after
infection
(c)
#
M.tuberculosis
CFU Assay
infection i.t. CD4+ and CD4+Foxp3+
analysis
DNA-hsp 65 i.m.
DNA-hsp 65 i.m.
CFP/CpG s.c.
pVAX i.m.
100
Day 90
80
Total number of lung
CD4+ cells (×104)
Experimental protocol
(a)
Controls
Saline
#
60
40
20
Non-immunized, infected mice
Non-immunized, uninfected mice
0
Lung
Spleen
6
*
*
5
&
*
5
*
&
4
&
&
4
3
4
2
CFU number/lung (Log10)
8
G
0·6
9
7
3
4
5
6
7
8
FC
G
G
BC
AX
/P
A-
pV
-C
65
p
65
hs
p
0·0
N
A-
hs
0·2
pG
5
0·4
BC
7
0·8
11
N
0
1·0
13
G
5
DNA-hsp 65
BCG/DNA-hsp 65
15
D
Total number CD4+Foxp3+
(×105)
10
6
(e)
/D
/C
G
BC
15
5
BC
pV
FP
hs
AN
/D
G
BC
AX
pG
-C
65
p
65
p
hs
AN
D
lung cells
20
Ratio of total number
CD4+/CD4+Foxp3+
6
0
Experimental
control groups
(g)
4
#
#
3
(f)
3
#
8
BC
CFU number/lung
(Log10)
6
CFU number/spleen
(Log10)
7
7
10
Total number of lung
CD4+Foxp3+ cells (×104)
8
8
Ratio CD4+/CD4+Foxp3+
lung cells (Total number)
(b)
(d)
Experimental
control groups
CFU number/lung (Log10)
Figure 2. Number of colony-forming units (CFU) in the lung and spleen and correlations of CFU, CD4+/CD4+ Foxp3+ ratio and CD4+ Foxp3+
cells from immunized mice. Different groups of mice were challenged with 1 9 105 bacilli of Mycobacterium tuberculosis 30 days after immunization (a). Lung and spleen CFU counts were evaluated on day 30 post challenge (b). The total number of lung CD4+ (c) and total number of lung
CD4+ Foxp3+ cells (d) are shown. The ratio of CD4+/CD4+ Foxp3+ cells (e), the ratio of lung CFU counts and CD4+/CD4+ Foxp3+ cells (f), the
ratio of lung CFU counts and total number of CD4+Foxp3+ cells (g) are shown. The cell number is expressed as the mean ± SEM for individual
animals. The CFU counts are expressed as Log10 of the mean ± SEM for individual animals. The data are representative of two independent
experiments (DNA-heat-schock protein (hsp) 65, 7; BCG/DNA-hsp 65, 7; BCG/CFP-CpG, 8; pVAX, 4; BCG, 7). *P < 005 compared with nonimmunized and infected mice (black dotted line); &P < 005 compared with non-immunized and infected mice (grey dotted line); #P < 005
compared with non-immunized, uninfected mice (solid line). Horizontal bars represent the significant difference between groups.
after antigen injection, or antigen and regulatory T cell
administration, footpad oedema was measured. We
observed that co-injection of Mtb antigen and regulatory
T cells isolated from uninfected or infected mice
suppressed oedema development. In addition, regulatory
T cells from infected mice were more suppressive than
regulatory T cells obtained from uninfected mice
(Fig. 4b). Next, we performed a DTH assay with spleen
244
CD4+ CD25+ cells that were obtained from nonimmunized, DNA-hsp 65-immunized or BCG/DNA-hsp
65-immunized mice (Fig. 4c). We found that the administration of the Mtb antigen in the presence of regulatory
T cells obtained from the DNA-hsp 65-immunized mice
(Mtb Ag + DNA-hsp 65 Treg) displayed a tendency,
although not significant, to decrease the footpad diameter
when compared with the footpad oedema that was a result
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
Heterologous vaccination against tuberculosis and CD4+Foxp3+ cells
Uninfected
Figure 3. Histological analysis of lungs. Mice
were vaccinated with DNA-heat-shock protein
(hsp) 65, BCG/DNA-hsp 65 or BCG/CFP-CpG
and challenged with Mycobacteirum tuberculosis
as described in Fig. 2. Pathology was evaluated
on the upper right lobe of the lung on day 30
post-challenge (original magnification, 100 9).
Asterisks show the areas represented in 400 9
magnification (upper right quadrant). Arrow
shows a foamy macrophage. Head arrow shows
neutrophils.
Infected
BCG/CFP-CpG
of the administration of Mtb antigen in the absence of
regulatory T cells (Mtb Ag; Fig. 4d). The footpad diameter
for mice that received Mtb antigen or Mtb antigen plus
regulatory T cells that were obtained from saline-injected
mice (Mtb Ag + Saline Treg) or mice that received Mtb
antigen plus regulatory T cells obtained from the BCG/
DNA-hsp 65 (Mtb Ag + BCG/DNA-hsp 65 Treg)-vaccinated mice were similar (Fig. 4d). There was no difference
in the footpad diameters after 48 hr (Fig. 4d).
A second assay that was used to deplete CD25+ cells
using mAbs against CD25 (PC61) was employed to evaluate the role of the regulatory T cells induced by vaccination. Figure 4(e) shows a schematic of the experimental
protocol used to deplete CD25+ cells before and during
the immunization schedule. Figure 4(f) shows that treatment with the mAb PC61 reduced the frequency of
CD4+ CD25+ cells in the peripheral blood. We observed
a significant reduction in the number of CFU in the lungs
of non-immunized, infected mice that were previously
treated with PC61 compared with non-immunized,
infected mice treated with a control antibody (Ig control;
Fig. 4g). Therefore, depletion of CD25+ cells contributed
to a reduction in the severity of infection. As expected,
both DNA-hsp 65 and BCG/DNA-hsp 65-immunized
mice previously treated with the control antibody were
significantly protected, as confirmed by the significant
reduction in the number of CFU compared with the
untreated, non-immunized and infected groups. Although
the treatment reduced the CFU numbers in the lungs
compared with the non-immunized, infected mice, it was
not different compared with the CFU counts in the lungs
of treated and untreated BCG/DNA-hsp 65-immunized
mice (Fig. 4g). These results suggest that the protection
conferred by the BCG/DNA-hsp 65 vaccine was not
dependent on CD25+ cells. It is likely that this vaccine
stimulated the differentiation of IFN-c-producing CD4+
cells more than CD4+ Foxp3+ cells.
Discussion
In this study, we demonstrated that heterologous immunization with BCG/DNA-hsp 65 or BCG/CFP-CpG was
BCG/DNA-hsp 65
DNA-hsp 65
pVAX
BCG
significantly more protective than DNA-hsp 65 immunization which induced a higher number of spleen
CD4+ Foxp3+ cells.
The induction of regulatory T cells is an important feature of the immune response that should be addressed
during the development of new vaccines against tuberculosis and other diseases. The induction of a high number
or increased frequency of regulatory T cells during immunization will help to drive the expansion of these cells
after an antigen is encountered or during infection with a
pathogen. This process can interfere with the efficacy of
the protective immune response. According to previous
studies that evaluated the number and function of regulatory CD4+ CD25+ or CD4+ Foxp3+ cells obtained from
the peripheral blood of patients with tuberculosis, these
cells contribute to the increased progression of infection.
This failure to control infection has been associated with
regulatory T cells inhibiting IFN-c secretion from effector
cells.19,20,31 Therefore, pre-clinical assays used in the
development of new vaccines against tuberculosis must
take into account the differentiation and activation of all
regulatory T cells, not only CD4+ Foxp3+ cells, but also
regulatory T1 cells and Th3 cells. Additionally, immunization schedules need to be designed dependent on the type
of immunization (homologous versus heterologous) and
the number of doses to avoid the expansion of higher
numbers of regulatory T cells that could affect the protective efficacy of the vaccination.
Heterologous immunization seems to represent an
advantage over homologous immunization. Modifying
the mechanism of release of the antigen to the immune
system reinforces the induction/expansion of a highly specific antigen immune response.32,33 BCG prime may be a
promising strategy because this vaccine is safe and
induces no side-effects. Among the various research
groups that study tuberculosis, there is a tendency to
maintain the use of the BCG vaccine and to improve its
immunogenic potential.33–35 Whole BCG bacteria may
contribute to the induction of the inflammatory response
and cytokine secretion that drives the differentiation of
the cellular immune response. The boost with CFP plus
CpG or DNA-hsp 65 could expand antigen-specific
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
245
P. F. Fedatto et al.
(a)
(c) Experimental protocol
Experimental protocol
Day 0
Hours
24 48
Day 30
Purification of CD4+CD25+ cells
from uninfected or 30 days
infected mice
M.tuberculosis
infection i.t.
Footpad
assessments
Footpad
assessments
Footpad
24 hr
48 hr
(d) 0·5
0·5
∆ DTH (mm)
0·4
∆ DTH (mm)
M.tuberculosis antigen in the
presence or absence of Treg cells
from immunized or non-immunized
mice
M.tuberculosis
infection i.t.
Administration of Mtb Ag or Mtb Ag plus Treg
in the footpad of 30 day infected mice
(b)
Hours
24 48
Day 30
Day 0
0·3
0·4
0·3
0·2
0·2
0·1
Mtb Ag
+
Mtb Treg
Saline Treg
+
–
–
+
+
–
Mtb
+
g
+S
(e)
Experimental protocal
Day 13
Day 15
Saline
BCG s.c.
Saline
DNA-HSP65 i.m.
PC61
Q4
102
103
104
104 105
Q3
CD4 PerCP-A
Q2
102 103
104 105
CD4 PerCP-A
102 103
CD4
Q1
0·8%
Q1
Q2
Q3
Q4
10
105
2
10
3
CD25
DNA-hsp 65
BCG/DNA-hsp 65
(g)
6·0
*
*
5·5
5·0
*
#
4·5
4·0
4
10
CD25 PE-A
CD25 PE-A
Mt
CFU Assay
PC61
3·5%
C
+B
6·5
PC61
Isotype Ab
(f)
g
bA
Tr
A
DN
G/
65
Non-immunized
Day 76
M.tuberculosis
infection i.t.
PC61
eg
p
-hs
30 days infection
Day 43
Day 46
CFU number/lung
(Log10)
Day –2
Day 0
-h
NA
bA
30 days after
immunization
5T
6
sp
D
g+
Mt
g
re
eT
n
ali
bA
Mt
–
reg
Ag
5
10
Ig Control
+
+
+
–
–
–
mAb PC61
(anti-CD25)
–
–
–
+
+
+
Figure 4. Evaluation of the regulatory T cells in vivo after immunization with the DNA-heat-shock protein (hsp) 65 or BCG/DNA-hsp 65 vaccine. BALB/c mice were infected intratracheally with 1 9 105 bacilli. Thirty days after infection, 50 lg Mycobacterium tuberculosis antigen (Mtb
Ag) in the presence or absence of CD4+ CD25+ cells, which were obtained from infected or uninfected mice, were administered in the right footpad. The left footpad received saline (a). Oedema measurement was performed 48 hr after the administration of antigen (b). BALB/c mice were
infected, and 30 days after infection, they received 50 lg M. tuberculosis antigens in the right footpad in the presence or absence of CD4+ CD25+
cells that were obtained from immunized or non-immunized mice (c). Oedema measurements were performed at 24 and 48 hr (d) after antigen
administration. BALB/c mice were immunized with BCG/DNA-hsp 65 and challenged 30 days after prime boost vaccination. Three days before
immunization and challenge, the mice were treated with the monoclonal antibody (mAb) PC61 (e). Representative flow cytometry data demonstrating the percentages of CD4+ CD25+ lymphocytes in the blood of mice treated with 500 lg mAb PC61 during immunization (f). Colonyforming unit (CFU) counts were evaluated 30 days after challenge (g). The results are expressed as the mean ± SEM. The data are representative
of two experiments. For the delayed type hypersensitivity (DTH) assay, with CD4+ CD25+ cells purified from uninfected or infected mice, seven
mice were used. For the DTH assay, with CD4+ CD25+ cells purified from immunized or non-immunized mice, 8–14 mice were used. For the
treatment with anti-CD25, eight or nine mice were used. Horizontal bars represent the significant difference between the groups. *P < 005 compared with non-immunized, infected and treated with immunoglobulin control mice; #P < 005 compared with mice non-immunized, infected
and treated with mAb PC61.
246
ª 2012 The Authors. Immunology ª 2012 Blackwell Publishing Ltd, Immunology, 137, 239–248
Heterologous vaccination against tuberculosis and CD4+Foxp3+ cells
lymphocytes that can control infection after the M. tuberculosis challenge. This is an important issue to be
addressed because we have previously shown that homologous vaccination with three doses of CFP plus CpG did
not protected immunized mice; in spite of intense IFN-c
production, there was severe lung injury.29 Although protective, high levels of IFN-c production need to be counterbalanced to minimize tissue inflammation and damage.
Regulatory T cells, IL-10 and TGF-b have all been associated with the inhibition of the cellular immune response
and inflammation. Heat-shock proteins are considered
highly immunogenic antigens, but several studies have
shown that these proteins have regulatory properties in
autoimmune diseases.36,37 Previously, we treated mice
with arthritis or diabetes with the DNA-hsp 65 construct
and observed that the treatment impaired the progression
of experimental disease. In addition, we found increased
staining for CD25 in the pancreas of treated mice, suggesting that the DNA-hsp 65 vaccine could induce T regulatory cells in the pancreas.36,37 Therefore, the induction
of a regulatory T-cell response may be important to avoid
the tissue damage that characterizes chronic inflammatory
diseases. Achievement of the proper ratio of effector
(CD4+) and regulatory (CD4+ Foxp3+) cells during the
immunization regimen will be challenging.
The first study to show that regulatory T cells could
influence the immunogenicity and protective efficacy of a
vaccine was carried out using a herpes simplex virus vaccine. In this study, Toka et al.38 demonstrated that the
depletion of CD25+ cells by the administration of an
anti-CD25 mAb (PC61) before DNA vaccine immunization increased the specific CD8+ T-cell response and
decreased the viral load better than immunized mice treated with a control antibody. An increase in the antigenspecific CD8+ cells in CD25-depleted mice was observed
in mice immunized with a DNA vaccine against hepatitis
B virus and in mice vaccinated against human papillomavirus.39,40 In tuberculosis, the role of T regulatory cells
induced by immunization has been evaluated with the
BCG vaccine. Jaron et al.41 demonstrated that after BCG
immunization, regulatory T cells were recruited to the
lymph nodes. The depletion of CD25+ cells with mAb
PC61 increased the production of IFN-c and slightly
reduced the CFU counts in the lungs compared with
untreated mice. On the contrary, Quinn et al.42 described
that the treatment with mAb against CD25 before BCG
immunization did not protect mice against M. tuberculosis challenge. These results indicate that the role of regulatory T cells during BCG immunization is still
controversial and needs to be investigated further. In this
study, we used the BCG vaccine as an experimental
control. Our efforts were focused on the DNA-hsp 65,
BCG/DNA-hsp 65 and BCG/CFP-CpG vaccines against
tuberculosis. The frequency, number and role of regulatory T cells in mice immunized with these vaccines have
not been evaluated previously. Prime-boost regimens
(BCG/DNA-hsp 65 and BCG/CFP-CpG) induced a higher
ratio of CD4+/CD4+ Foxp3+ cells in the spleen compared
with non-vaccinated mice and BCG-vaccinated mice. In
addition, the positive correlation between the number of
regulatory T cells and CFU counts suggests that the
protective efficacy of vaccines against tuberculosis is associated with a decreased number of CD4+ Foxp3+ cells.
Although we observed that CD4+ CD25+ cells transferred from DNA-hsp 65-immunized mice had a tendency
to suppress the DTH response compared with
CD4+ CD25+ cells transferred from BCG/DNA-hsp 65immunized mice or from mice that did not receive regulatory T cells, these differences were not significant. In
addition, although the depletion of CD25+ cells from nonimmunized mice induced a significant reduction in the
lung CFU count compared with non-immunized mice
treated with a control antibody, the depletion of CD25+
cells did not affect the protective efficacy of the BCG/
DNA-hsp 65 immunization. This result is probably associated with the increased ratio of CD4+/CD4+ Foxp3+ cells
in the spleen that was induced after vaccination and in the
lungs after challenge. This leads to two hypotheses: (i) the
BCG/DNA-hsp 65 vaccine is a weak inducer of regulatory
T cells or (ii) this vaccine is a strong inducer of CD4+ cells
and the ratio between these two cell types counterbalances
the protection and lung injury.
Finally, these results confirm the protective efficacy of
the prime-boost vaccines, BCG/DNA-hsp 65 and BCG/
CFP-CpG, and the DNA-hsp 65 homologous vaccine.
They also indicate that these prime-boost regimens are
promising strategies for the development of a new vaccine
against tuberculosis because they induce an effective cellular immune response that is associated with a reduced
number of regulatory cells and decreased lung injury.
Acknowledgements
We would like to thank Fabiana Rossetto de Morais for
help with flow cytometry; Rita de Cassia Aguiar Tinasi
for BCG donation; and Izaira Tincani Brandão and Ana
Paula Masson for general technical assistance. This work
was supported by the Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) grant 2007/02695-5.
Disclosures
The authors declare no conflict of interest.
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