Carvalho 2008

Immune cellular parameters of leprosy and human
immunodeficiency virus-1 co-infected subjects
Introduction
Leprosy is a chronic infectious disease, affecting the skin
and peripheral nerves, caused by the intracellular bacillus
Mycobacterium leprae.1
The incidence of new cases of lep-
rosy remains constant at 286 000 per year, and Brazil is
one of the countries worst affected, accounting for the
majority of new cases reported in the Americas.2
As the
prevalence rates of human immunodeficiency virus-1
(HIV-1) infection are escalating in some countries where
leprosy is endemic, one might expect that the geographic
overlap of the two epidemics may lead to increased
numbers of co-infected patients. The current situation
concerning leprosy endemicity and HIV-1 prevalence in
Brazil and other countries emphasizes the importance of
monitoring for co-infections.3
In addition to the public
health aspect of this co-infection, these pathogens may
have a potentially interesting immunologic interaction in
the human host.
It has been previously suggested that leprosy is a
human infection model in which to study the T helper
1/T helper 2 (Th1/Th2) paradigm,4
permitting the delin-
eation of polarized human T helper responses in response
to a single pathogen. The spectrum of M. leprae-specific
immune responses between these poles correlates with the
range of clinical manifestations of the infection.5
At the
Karina I. Carvalho,1
Solange
Maeda,1
Luciana Marti,2
Jane
Yamashita,1
Patrick A. J. Haslett3
and Esper G. Kallas1
1
Federal University of Saõ Paulo, Saõ Paulo,
Brazil, 2
Albert Einstein Research Institute, Saõ
Paulo, Brazil, and 3
University of Miami, FL,
USA
doi:10.1111/j.1365-2567.2007.02756.x
Received 1 June 2007; revised 15 October
2007; accepted 16 October 2007.
Correspondence: E. G. Kallas, MD, PhD,
Laborato´rio de Imunologia, Disciplina de
Doenças Infecciosas e Parasita´rias, Escola
Paulista de Medicina/UNIFESP, Rua Mirassol
207, 04044-010 - Saõ Paulo – SP, Brazil.
Email: kallas.dmed@epm.br
Senior author: Esper Kallas
Abstract
Leprosy and human immunodeficiency virus-1 (HIV-1) are examples of
human infections where interactions between the pathogen and the host
cellular immunity determine the clinical manifestations of disease. Hence,
a significant immunopathological interaction between HIV-1 and leprosy
might be expected. In the present study we explored several aspects of cel-
lular immunity in patients co-infected with HIV-1 and Mycobacterium
leprae. Twenty-eight individuals were studied, comprising four groups:
healthy controls, HIV-1 and M. leprae co-infection, HIV-1 mono-infec-
tion, and M. leprae mono-infection. Subjects in the mono-infection and
co-infection groups were matched as far as possible for bacillary load and
HIV disease status, as appropriate. Peripheral blood mononuclear cells
(PBMC) were analysed using six- and seven-colour flow cytometry to
evaluate T-cell subpopulations and their activation status, dendritic cell
(DC) distribution phenotypes and expression of IL-4 by T cells. The
co-infected group exhibited lower CD4 : CD8 ratios, higher levels of
CD8+
T-cell activation, increased Vd1 : Vd2 T cell ratios and decreased
percentages of plasmacytoid DC, compared with HIV-1 mono-infected
subjects. Across infected groups, IL-4 production by CD4+
T lymphocytes
was positively correlated with the percentage of effector memory CD4+
T
cells, suggesting antigenically driven differentiation of this population of
T cells in both HIV-1 and M. leprae infections. Co-infection with M. le-
prae may exacerbate the immunopathology of HIV-1 disease. A T helper
2 (Th2) bias in the CD4+
T-cell response was evident in both HIV-1
infection and leprosy, but no additive effect was apparent in co-infected
patients.
Keywords: HIV; leprosy; co-infection; lymphocytes; IL-4
206 Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214
IMMUNOLOGY ORIGINAL ARTICLE

Th1 pole, tuberculoid, or paucibacillary, leprosy is
characterized by high levels of specific cell-mediated
immunity that effectively limits bacillary replication and
is associated with limited disease, although often with
concomitant immunological damage to the nerves. At the
other pole, lepromatous, or multibacillary, leprosy is
characterized by a selective unresponsiveness to M. leprae
antigens, diffuse cutaneous disease and the uncontrolled
multiplication of organisms in the skin, often to extra-
ordinary numbers. Much of the morbidity of leprosy
results from episodic inflammatory exacerbations of lep-
rosy lesions in the skin and nerves, called ‘lepra’ reac-
tions, thought to be caused by spontaneous shifts in host
immunity.6
Because HIV-1 infection has a profound effect on the
incidence and clinico–pathological features of other myco-
bacterial diseases, such as tuberculosis, one might expect a
significant interaction also to exist between HIV-1 and
leprosy.7
In HIV-1/M. tuberculosis co-infections, immune
suppression secondary to HIV-1 infection accelerates the
progress of tuberculosis, and, conversely, the cellular
immune activation associated with tuberculosis is associ-
ated with more rapid progression of HIV-1 disease.8,9
In
the setting of HIV-1/M. leprae co-infections, there has
been a general expectation that immune deficiency caused
by HIV-1 infection would shift the spectrum of leprosy
towards the lepromatous (Th2) pole, although epidemio-
logical data are sparse and conflicting.10
Paradoxically, the
most detailed description of leprosy immunopathology in
HIV-1 co-infected patients revealed no change in immune
cell infiltrates across the leprosy spectrum, despite
advanced HIV-1-associated immune deficiency.7,11
On the
other hand, there has been little or no attempt to evaluate
the impact of M. leprae infection on HIV-1 pathogenesis.
This interaction has been studied in the macaque/simian
immunodeficiency virus (SIV) model, however, where
M. leprae infection was observed to exert an unexpected
and unexplained ameliorating effect on SIV disease, pro-
longing survival of the animals, despite equal or increased
viral burdens.12
In light of these various reported interactions of myco-
bacterial infections with HIV and SIV pathogenesis, we
were interested in investigating whether human M. leprae
co-infection might exacerbate or attenuate HIV-1 patho-
genesis. As an initial exploration of these questions, we
performed a cross-sectional analysis of immune cellu-
lar parameters in blood cells from relatively rare HIV-1
and M. leprae co-infected subjects, in comparison with
HIV-1 and M. leprae mono-infected subjects, and healthy
volunteers.
Our investigation focused on peripheral blood immune
cells that are known to be altered in HIV-1 disease and
that are implicated in the immunity and/or pathogenesis
of mycobacterial infections, including leprosy. Thus, in
addition to CD4 and CD8 T-cell subsets, we examined
the two main subsets of cd T cells: Vd2 cells and
Vd1 cells. Vd2 cells are stimulated by isoprenoid
phosphoantigens that are present in bacteria, including
mycobacteria.13
This population of cells plays a role in
antimycobacterial defense,14,15
but the cell population
shrinks dramatically during acute HIV-1 infection, with
variable recovery following antiviral chemotherapy.16,17
In
contrast, the Vd1 population, of unknown function,
expands during HIV-1 infection, so that the ratios of Vd1
to Vd2 T cells are increased with progressive HIV-1 infec-
tion.16,17
We also examined the two main subsets of
peripheral blood dendritic cells (DC), called plasmacytoid
and myeloid DC. DC are key components of the innate
immune system, acting as antigen-presenting cells that are
essential for the priming and regulation of T-cell immu-
nity. Hence, the responses and interactions of these popu-
lations of DC are thought to determine whether T cells
differentiate into Th1 or Th2 cells,18
spanning the range
of phenotypes observed in leprosy. Mycobacteria are
known to stimulate DC via toll-like receptors (TLR) pres-
ent on both myeloid (TLR2) and plasmacytoid (TLR9)
subsets.19–21
Both subsets of DC can be infected by HIV-1,
but a differential and striking loss of peripheral blood
plasmacytoid DC characterizes progressive HIV-1 dis-
ease.22
In light of the complex and contrasting effects of
HIV-1 and mycobacterial infections on cd T-cell and DC
populations, we were interested in examining these
immune cells in patients with HIV-1 and M. leprae
co-infections.
Materials and methods
Subjects and sample collection
This study was reviewed and approved by the local institu-
tional review board (IRB, Comiteˆ de E´tica em Pesquisa
Humana da Universidade Federal de Saõ Paulo/UNIFESP),
and IRB-approved informed consent was obtained from
all participants. Leprosy patients were treated according to
World Health Organization guidelines.23
Acquired immu-
nodeficiency syndrome (AIDS) was defined using modified
criteria adopted by the Brazilian Ministry of Health that
includes patients with a CD4 cell count of < 200 cells/ll
or clinical conditions related to AIDS.24
Seven healthy controls and seven HIV-seropositive
patients, most of whom had CD4+
T-cell counts of < 400
cells/ll, were identified at UNIFESP. Seven patients with
leprosy were enrolled at the Leprosy Clinic at the State
Health Department (Sao Paulo, Brazil) and were classified
according to their bacillary load.25
Seven patients
co-infected with leprosy and HIV-1 infection were
recruited at UNIFESP, using local identification and refer-
ral from other services in Sao Paulo. Leprosy patients
were matched for bacillary load with the patients in the
co-infected group. In this study, the major presentations
Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Publishing Ltd, Immunology, 124, 206–214 207
Immunity in M. leprae and HIV-1 co-infection

of leprosy were the paucibacillary form rather than the
multibacillary form.
The HIV mono-infected and co-infected patients were
receiving highly active antiretroviral therapy (HAART)
and multidrug therapy (MDT). Patients with immune
reconstitution inflammatory syndrome were not included
in the present study to avoid potential interference in the
immune parameters, as described in different mycobacte-
rial diseases.26–28
Peripheral blood mononuclear cells (PBMC) were iso-
lated from the study subjects by density-gradient sedi-
mentation over Ficoll–Paque (Pharmacia Biotech,
Uppsala, Sweden). The isolated PBMC were then washed
twice in Hank’s balanced salt solution (Gibco, Grand
Island, NY). Cells were cryopreserved in RPMI 1640
(Gibco), supplemented with 20% heat-inactivated fetal
bovine serum (FBS; HyClone Laboratories, Logan, UT),
50 U/ml of penicillin (Gibco), 50 lg/ml of streptomycin
(Gibco), 10 mM glutamine (Gibco) and 7Á5% dimethyl
sulphoxide (DMSO; Sigma, St Louis, MO). Cryopre-
served cells were stored in liquid nitrogen until used in
the assays. At the time of the assay, PBMC were rapidly
thawed in a 37° water bath and washed in RPMI 1640
supplemented with 10% fetal calf serum, 100 U/ml of
penicillin, 100 lg/ml of streptomycin and 20 mM
glutamine (R10). Cells were counted, checked for viabil-
ity and resuspended in R10 at a concentration of 106
cells/ml.
Plasma HIV-1 RNA detection
The plasma HIV RNA detection load was assessed using
the ultrasensitive AMPLICOR HIV-1 MONITOR test ver-
sion 1.5 (Roche Diagnostics, Indianapolis, IN), according
to the manufacturer’s instructions.
Flow cytometry
The following monoclonal antibodies were used for
surface staining: CD3–allophycocyanin (APC) (clone
UCHT1), CD8–allophycocyanin carbocyanin 7 (APC-
Cy7) (clone SK1), Vd2–phycoerythrin (PE) (cloneB6),
CD45RA–peridin chlorophyll protein (PerCP) (clone HI
100), CCR7–phycoerithrin carbocyanin 7 (PeCY7) (clone
3D12) and CD69–fluorescein isothiocyanate (FITC)
(clone FN50), from BD PharMingen (San Jose, CA);
CD4–Alexa 610 (clone S3.5) from Caltag Laboratories
(Burlingame, CA); Vd1–FITC (clone T58.2) from Endo-
gen (Rockford, IL); Lineage Cocktail 1 (Lin 1: CD3,
CD14, CD16, CD19, CD20 and CD56) FITC, human
leucocyte antigen (HLA)-DR–PerCP (clone L243),
CD11c–APC (clone S-HCL3), CD123–PE [anti-interleukin
(IL)-3 receptor], CD38–PE (clone HB7), CD4–FITC (clone
L120), from BD Biosciences (San Jose, CA); and CD25–
PE–CY7 (clone BC96), from e-Bioscience, (San Diego,
CA). Intracellular staining for cytokines was performed
using mouse anti-human IL-4–PE (clone 3010.211), mouse
anti-human interferon (IFN)-c–PE–CY7 (clone B27) and
mouse anti-human tumour necrosis factor (TNF)-a–APC
(clone Mab11), all from BD PharMingen. Fluoresce minus
one (FMO) was used for gate strategy.29
In some experiments, thawed PBMC were incubated in
24-well plates (1 ml/well) (Becton Dickinson, San Jose,
CA) in the presence of 1 lM ionomycin (Sigma) and
20 ng/ml of phorbol 12-myristate 13-acetate (PMA;
Sigma), for 16 hr. After stimulation, cells were centrifuged
at 1500 g for 5 min and transferred into V-bottom
96-well plates (Nunc, Roskilde, Denmark) in 100 ll of
staining buffer [phosphate-buffered saline (PBS) supple-
mented with 0Á1% sodium azide (Sigma) and 1% FBS,
pH 7Á4–7Á6] with the panel of surface monoclonal anti-
bodies. Cells were incubated at 4° in darkness for 30 min,
washed twice and then resuspended in 100 ll of fixation
buffer [1% paraformaldehyde (Polysciences, Warrington,
PA) in PBS, pH 7Á4–7Á6].
Intracellular staining was performed after surface stain-
ing with CD4–FITC, CD3–PerCP and CD8–APC–CY7.
Cells were incubated with 100 ll of 4% fixation buffer
and washed with permeabilization buffer (PBS supple-
mented with 0Á1% sodium azide, 1% FBS and 0Á1% sapo-
nin; Sigma). Each sample was resuspended in 100 ll of
permeabilization buffer, incubated for 15 min at room
temperature in the dark, washed with 100 ll of staining
buffer and incubated for 30 min at 4° in the dark with
either no antibody (unstained tube) or anti-IL-4–PE, anti-
IFN-c–PE–CY7 and anti-TNF-a–APC in 50 ll of staining
buffer.30
Cells were washed with 200 ll of staining buffer
and resuspended in 100 ll of 1% paraformaldehyde (PFA)
for flow cytometry analysis. Samples were acquired on a
FACSCanto or FACSAria, using FACSDIVA software (BD
Biosciences), and the analysed with FLOWJO software (Tree
Star, San Carlo, CA). Fluorescence voltages were deter-
mined using matched unstained cells. Compensation was
carried out using CompBeads (BD Biosciences) single-
stained with CD3–PerCP, CD4–FITC, CD8–APC–CY7,
CD4–PE–CY7, CD3–PE or CD3–APC. Samples were
acquired until at least 200 000 events in a live lymphocyte
gate or at least 500 000 events in a live DC gate were
obtained.
Statistical analyses
Groups were compared using non-parametric models;
data are reported as median and interquartile range.
Comparisons among groups were carried out using the
Kruskall–Wallis non-parametric test, followed by inter-
group comparisons by the Dunnet test. Correlations were
performed using the Spearman non-parametric test.
P-values were considered significant if <0Á05. Results are
expressed in medians and interquartile ranges (IQR).
K. I. Carvalho et al.

Results
Characteristics of the HIV-1-M. leprae co-infected
patients
Demographic, clinical, microbiological and laboratory
characteristics are detailed in Table 1. The median ages
of all participants was 38 years (IQR: 35–48) and most
were male (83Á9%). No difference in gender distribution
was observed between groups. For the leprosy and co-
infected groups, 80% of the patients had a paucibacil-
lary presentation at the time of diagnosis. Co-infected
patients were treated with the appropriate MDT for
paucibacillary and multibacillary leprosy. Median CD4+
T-cell counts in both HIV-infected groups were
matched (949 cells/ll, IQR 727Á5–1465 for controls; 297
cells/ll, IQR 265–410 for HIV-infected patients; and
236 cells/ll, IQR 161–390 for co-infected patients
Table 1).
Leprosy and HIV-1 infections lead to marked
disturbances of T-lymphocyte distribution
The CD4 : CD8 T-cell ratio was decreased in both HIV-1-
infected groups, but more in co-infected patients
compared with controls (0Á16, IQR 0Á09–0Á20; and
1Á30, IQR 0Á95–1Á9, respectively, P < 0Á001, Fig. 1a).
Although not statistically significant, co-infected patients
exhibited lower CD4 : CD8 ratios than HIV-1 mono-
infected subjects, suggesting more severe immunopathol-
ogy in the former group, despite similar CD4+
T-cell
counts (Fig. 1a).
Table 1. Demographic, clinical and laboratory characteristics of participants
Case
numbers1
Groups Gender
Age
(years)
Leprosy
clinical
presentation
Bacillary
index
Leprosy
therapy
(months
of MDT)
Viral load
(HIV-RNA
copies/ml)
CD4+
T cells/
mm3
HIV
therapy
102 Control Male 29 – – – – 1358 –
103 Control Female 47 – – – – 1695 –
104 Control Male 40 – – – – 742 –
105 Control Male 34 – – – – 774 –
106 Control Male 49 – – – – 1084 –
110 Control Male 54 – – – – 661 –
113 Control Female 51 – – – – 949 –
128 Control Male 37 – – – – 1571 –
131 Control Male 38 – – – – 713 –
142 Control Male 49 – – – – 980 –
1001 HIV Male 37 – – – <399 405 HAART
1004 HIV Male 34 – – – 925 503 HAART
1020 HIV Male 33 – – – <399 410 HAART
1039 HIV Male 35 – – – 200 170 HAART
1050 HIV Male 51 – – – <399 265 HAART
2008 HIV Male 38 – – – <399 275 HAART
2011 HIV Male 38 – – – 762 297 HAART
1 HIV-Leprosy Male 38 BL 2+ 10 <399 161 HAART
2 HIV-Leprosy Male 38 BT Negative 12 <399 269 HAART
3 HIV-Leprosy Male 31 BT 1+ 8 <399 235 HAART
4 HIV-Leprosy Female 51 BL 1+ 12 <399 390 HAART
5 HIV-Leprosy Male 35 BT Negative 12 7220 127 HAART
6 HIV-Leprosy Male 53 BT Negative 2 <399 236 HAART
7 HIV-Leprosy Male 47 BT 1+ 5 <399 481 HAART
10 Leprosy Male 38 LL 3+ 20 – ND
11 Leprosy Male 43 TT Negative 4 – ND
12 Leprosy Male 37 BT Negative 7 – ND
13 Leprosy Male 33 BT Negative 5 – ND
15 Leprosy Female 31 BL 1+ 14 – ND
BL, borderline-lepromatous; BT, borderline-tuberculoid; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; LL,
lepromatous-lepromatous; MDT, multidrug therapy; ND, not done; TT, tuberculoid.
1
Case numbers reflect the enrollment sequences only within each individual group.

Surface activation markers were evaluated in all four
groups. Marked HLA-DR up-regulation on CD8+
T cells
was observed in both HIV/M. leprae co-infected and HIV
mono-infected groups compared with controls [mean flu-
orescence intensities of 165 (IQR 94–271), 134 (IQR 114–
249) and 46 (IQR 21–68), respectively, P < 0Á05, Fig. 1b],
but this was not seen in CD4+
T cells. There was a non-
significant trend towards increased HLA-DR expression in
the co-infected compared with the HIV-1 mono-infected
group (Fig. 1b). No differences were observed in the
expression of CD69, CD25 and CD38 on CD4+
and
CD8+
T lymphocytes (data not shown).
Dendritic cells and Vd2 T lymphocytes are
proportionately diminished in co-infected patients
The Vd2 T-cell subset was decreased in the co-infection
group when compared with the control group (median
1Á53%, IQR 0Á73–2Á4, P < 0Á05), whereas the percentage
of Vd1 T cells was similar in all groups. There was a sta-
tistically significant overall difference in the Vd1 : Vd2 cell
ratio, exaggeratedly inverted in the co-infected group
compared with subjects infected with HIV-1 only
(co-infection, 30Á3%, IQR 9Á9–35Á7; HIV, 5Á9%, IQR 3Á8–
13Á7; controls, 0Á63%, IQR 0Á25–3Á1; and leprosy, 2Á86%,
IQR 0Á44–10Á84, P < 0Á05, Fig. 1c).
The percentages of plasmacytoid DC in total PBMC
were diminished in co-infected patients when compared
with controls (co-infected, 0Á01%, IQR 0Á005–0Á02; HIV,
0Á02%, IQR 0Á005–0Á18; control, 0Á13%, IQR 0Á09–0Á18;
and leprosy, 0Á03%, IQR 0Á0–0Á15, P < 0Á05, Fig. 1d).
On the other hand, no significant differences in the
percentages of myeloid DC were observed (data not
shown).
HIV-1 and leprosy drives the maturation of
T lymphocytes
CD4+
T cells were stained for surface expression of
CD45RA and CCR7. Phenotypic nomenclature was based
on that proposed by Sallusto et al., where CCR7+
CD45RA+
are described as naı¨ve cells, CCR7+
CD45RA)
as central memory cells and CCR7)
CD45RA)
as effector
memory cells 31
. Control subjects had higher percentages
of naı¨ve and central memory cells compared with the
other groups, with a corresponding decrease in the pro-
portion of effector memory cells (Fig. 2). The most pro-
nounced difference in these maturation subsets was seen
when control subjects were compared with leprosy
patients (CCR7+
CD45RA+
naı¨ve: 5Á47%, IQR 1Á66–18Á2
for controls and 0Á56%, IQR 0Á22–1Á84 for leprosy;
CCR7+
CD45RA)
central memory: 33Á8%, IQR 28Á1–35Á7
for controls and 15Á05%, IQR 8Á6–22 for leprosy;
CCR7)
CD45RA)
effector: 49Á9%, IQR 47Á6–64 for con-
trols and 82Á6%, IQR 75Á15–87Á3 for leprosy). For CD8+
T-cell subsets, the only statistically significant difference
was observed when comparing central memory CCR7+
CD45RA)
cells from control subjects (17Á7%, IQR 13Á15–
30) with co-infected (4Á28%, IQR 2Á63–13Á2) and leprosy
(5Á67%, IQR 4Á79–10Á45) patients.
Both pathogens tend to direct the immune response
towards IL-4 production
Next, we assessed cytokine production after PMA and
ionomycin stimulation. No differences were observed in
the production of TNF-a and IFN-c between CD4+
and
CD8+
T lymphocytes. On the other hand, IL-4 produc-
tion, determined by high expression of IL-4 in gated
Control HIV HIV–leprosy Leprosy
0·0
0·5
1·0
1·5
2·0
2·5(a) (b)
(c) (d)
CD4:CD8ratioVδ1:Vδ2ratio
PlasmocytoidDC(%)HLA-DRMFI
P < 0·01
0
100
200
300
400
500 P < 0·01
0
10
20
30
40
50
60
70
P < 0·01
Control HIV HIV–leprosyLeprosy
0·0
0·1
0·2
0·3
0·4
0·5
0·6
0·7 P < 0·01
Figure 1. Several cellular immunological mark-
ers obtained using flow cytometry were evalu-
ated and compared among the four groups of
volunteers. These markers comprised (a) the
CD4 : CD8 ratio, (b) cellular activation of
CD8+
T cells, measured by human leuco-
cyte antigen (HLA)-DR expression, (c) the
Vd1 : Vd2 ratio and (d) the percentage of plas-
macytoid dendritic cells among total peripheral
blood mononuclear cells (PBMC). Compari-
sons were carried out using the Kruskal–Wallis
non-parametric test followed by intergroup
comparisons by the Dunnet test. HIV, human
immunodeficiency virus; MFI, mean fluores-
cence intensity.

CD4+
T cells, was statistically lower in controls (0Á57%,
IQR 0Á33–0Á93) when compared with the other three
groups (1Á09%, IQR 0Á62–2Á85; P = 0Á03). No statistically
Central memory
Naïve
Effector memory
0
10
20
30
40
P < 0·01
0
10
20
(a)
(b)
(c)
P < 0·01
45
50
55
60
65
70
75
80
85
90
95
P < 0·01
CCR7+
CD45RA–
%inCD4+
TcellsCCR7–
CD45RA–
%inCD4+
TcellsCCR7+
CD45RA+
%inCD4+
Tcells
Figure 2. Distribution of cellular maturation markers of CD4+
T
cells. Cellular subpopulations were determined by the expression of
CCR7 and CD45RA after gating on CD3+
CD4+
cells. The percent-
age of (a) naı¨ve (CCR7+
CD45RA+
), (b) central memory (CCR7+
CD45RA)
) and (c) effector memory (CCR7)
CD45RA)
) cells are
depicted for all groups of subjects. HIV, human immunodeficiency
virus.
0 5 10 15 20 25 30 35 40
0
1
2
3
4
5
6
7
(c)
(b)
(a)
CD4
+
TcellsproducingIL-4(%)
r = –0·5861; P = 0·0003
30 40 50 60 70 80 90 100
CCR7
–
CD45RA
–
among CD4
+
T cells (%)
CCR7
–
CD45RA
–
among CD4
+
T cells (%)
0
1
2
3
4
5
6
7
CD4
+
TcellsproducingIL-4(%)
r = 0·4791; P = 0·0041
Control Co-infected
SSC
IL-4
0 102
103
104
105
0
1000
2000
3000
4000
0·42%
0 102
103
104
105
4·25%
Figure 3. Interleukin-4 (IL-4) production was determined by intra-
cellular staining and flow cytometry after stimulation with iono-
mycin and phorbol 12-myristate 13-acetate for 16 hr. (a) The IL-4+
gate was set for cells producing high levels of cytokine. The level of
IL-4 production was negatively correlated with the percentage of
central memory (CCR7+
CD45RA)
) CD4+
T cells (b) and positively
correlated with effector memory (CCR7)
CD45RA)
) CD4+
T cells
(c). The results for all four study subject groups are shown: solid cir-
cles, healthy controls; open circles, co-infection; open triangles,
human immunodeficiency virus-1; open squares, leprosy. Correla-
tions were assessed using the non-parametric Spearman’s test. SSC,
side scatter.

significant differences of cytokine production by CD8+
T
cells were observed.
The IL-4 production by CD4+
T lymphocytes was nega-
tively correlated with the percentage of central memory
cells (r = )0Á59, P < 0Á01) and positively correlated with
the percentage of effector CD4+
T cells (r = 0Á48,
P < 0Á01) (Fig. 3). As shown in Fig. 3, the frequency of
IL-4+
T cells in control subjects clustered tightly, whereas
those in the three infected groups were increased and in
an overlapping distribution.
Discussion
There is considerable epidemiological overlap between
M. tuberculosis and HIV epidemics, so that co-infections
may be common in certain areas. In contrast, at present,
rather distinct populations tend to be infected with
M. leprae and HIV, so co-infections are much less com-
mon. However, future projections of spread of the HIV
epidemic into areas with more prevalent M. leprae infec-
tion may change the co-infection epidemiological char-
acteristics. The importance of tuberculosis and HIV
co-infection as a public health problem is obvious, but
this is less clear for M. leprae and HIV co-infections.32,33
However, the special nature of M. leprae stimulates
unique questions about the possible consequences of
co-infection. Infection with M. leprae differs in several
ways from that with M. tuberculosis – there is a much
more gradual evolution of disease, a classic spectrum of
clinical manifestations related to Th1 and Th2 responsive-
ness by the host, often huge antigenic burdens that are
slow to clear, and pathogenesis that is largely caused by
spontaneous shifts in host immune responsiveness, result-
ing in inflammatory lepra reactions.
We set out to compare cellular immune parameters in
HIV-1-infected patients with and without leprosy. A
limitation of the present study was the small sample size,
owing to the relative rarity of HIV-1/M. leprae co-in-
fected patients. Moreover, the challenge of interpreting
results from this cohort was compounded by the variabil-
ity of HIV disease, according to stage of progression,
superimposed on the spectral nature of leprosy. In an
attempt to derive meaningful data from the present
sample, we endeavoured to match HIV-1 and M. leprae
co-infected patients with HIV-1 and M. leprae mono-
infected subjects, for CD4 and bacillary index, respec-
tively (Table 1).
In the present study, we confirmed that leprosy
mono-infection is associated with increased IL-4 produc-
tion by CD4+
T cells (Fig. 3). A similar increase was
observed in HIV-1 mono-infection, as has been reported
by others,34
but no apparent additive or synergistic effect
was seen in HIV-1/M. leprae co-infected patients. Our
data suggest that leprosy co-infection may aggravate,
rather than ameliorate, HIV pathogenesis, as indicated
by the decreased ratio of CD4 : CD8 T cells, higher
frequency of activated CD8+
T cells and loss of plasma-
cytoid DC, all recognized features of progressive HIV-1
disease. This is in contrast to the observation of Gormus
et al., who made the unexpected observation of SIV
disease amelioration in the setting of experimental M. le-
prae co-infection of rhesus macaques.12
In the latter
studies, we speculate that the immunologic environment
associated with a high M. leprae antigenic burden might
have attenuated the immune activation-driven pathogen-
esis of SIV disease. However, our data do not support
the hypothesis that M. leprae co-infection can attenuate
the immunopathogenesis of human HIV-1 disease. On
the contrary, the results suggest that M. leprae co-infec-
tion may exacerbate HIV-1 pathogenesis. Clearly, there
are important differences between the macaque model
system and natural human infections. Macaques are
natural hosts of neither M. leprae nor SIV, and the ani-
mals were infected with a large intravenous inoculum of
bacilli. Perhaps most importantly, no inflammatory man-
ifestations of leprosy were described in the experimental
animals. On the other hand, inflammatory lepra
reactions can complicate up to half of human cases of
leprosy, and this immunopathology may indeed account
for much of the nerve damage and morbidity of this
disease. Cutaneous and systemic expression of pro-
inflammatory cytokines, such as TNF-a, have been
extensively documented in lepra reactions35,36
and may
be expected to promote HIV-1 replication. Indeed, cyto-
kine-driven enhancement of viral replication has been
invoked to explain the aggravation of HIV-1 disease in
patients with concurrent tuberculosis.37
Thus, in HIV-1/
M. leprae co-infection, inflammation associated with
clinical or subclinical lepra reactions may offset the
potential for any beneficial immune-modulatory effects
of M. leprae on HIV-1 disease progression.
Sallusto et al. described that immunological memory is
displayed by distinct T-cell subsets: lymph node-homing
CCR7+
CD45RA)
(central memory T cells, TCM) and tis-
sue-homing cells CCR7)
CD45RA)
(effector memory
T cells, TEM).31
Our results suggest that leprosy patients
have a decreased number of naı¨ve cells when compared
with healthy controls, together with a decreased percent-
age of TCM and an increased percentage of TEM, mostly
in co-infected patients. We hypothesize that the
imbalance in the percentage distribution seen in leprosy
and co-infected patients reflects a switch from naı¨ve to
memory CD4+
T lymphocytes, as a result of continuous
antigenic stimulation and cellular activation, as also seen
in the context of tuberculosis.38
This finding may well
represent a reactive expansion of ‘protective memory’
TEM cells in response to M. leprae and HIV as a result of
differentiation of TCM to combat the pathogen, especially
in the tissues, considering the high antigenic burden
observed in both diseases.39

Curiously, a positive correlation was observed between
the proportional expansion of circulating TEM CD4+
T
cells and the percentage of IL-4+
-producing CD4+
T cells
after stimulation with PMA and ionomycin (Fig. 3b).
Although our analysis did not permit us to ascertain
directly whether TEM are actually the producers of IL-4, it
is likely that these Th2-differentiated cells are indeed anti-
gen-experienced members of the CD4+
TEM population.
This interpretation is consistent with previous reports of
higher IL-4 production in the context of M. leprae40,41
and HIV-142
infections. As increased frequencies of these
cells were observed in chronic HIV-1 and/or M. leprae
infections, there is clearly an association between IL-4
production and the presence of antigen. However, our
approach did not address the antigen specificity of the
IL-4-producing T cells. Others have demonstrated expres-
sion of Th2 cytokines in leprosy lesions,35,40,43
which may
represent antigen-driven or cytokine-driven expansion of
M. leprae-specific T cells.44
These responses may be influ-
enced by the genetic background of the individual as well
as by environmental factors.44
We suggest that the contin-
uing production of IL-4 by HIV-1 and M. leprae-specific
T cells may create a ‘Th2 environment’ in which the
priming of T cells to heterologous antigens is biased
towards IL-4 production.45
Exploring the association of
higher IL-4 production after PMA and ionomycin stimu-
lation, and expansion of TEM, may present an opportu-
nity to elucidate the mechanisms involved in the possibly
deleterious effect of M. leprae infection in HIV-1-infected
patients observed in our study.
In conclusion, this initial exploration of the cellular
immune interactions of leprosy and HIV-1 disease sug-
gests that chronic infection with M. leprae might exacer-
bate the immunopathogenesis of HIV-1 disease. We
speculate that this may be the result of a combination of
inflammatory lepra reactions and the aggravated Th2
environment induced by M. leprae antigens. Prospective
longitudinal studies are needed to address the questions
raised in this work.
Acknowledgements
This work was partially supported by Fundaçaõ Paulista
contra a Hansenıáse, National Institutes of Health, grant
#R01-AI052731-06, and The Fogarty International Center,
grant #D43 TW00003; KCS’s PhD scholarship was pro-
vided by the Conselho Nacional de Desenvolvimento
Cientı´fico e Tecnolo´gico (CNPq), Brazilian Ministry of
Science and Technology. We are also thankful for support
from the Heiser Program for Research in Leprosy and
Tuberculosis of The New York Community Trust.
Conflicts of interests
The authors declare no competing conflicts of interests.
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