Therapeutic Enhancement of Protective Immunity during
Senad Divanovic1, Aurelien Trompette1¤, Jamie I. Ashworth1, Marepalli B. Rao2, Christopher L. Karp1*
1Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center, and the University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of
America, 2Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America
Background: Leishmaniasis remains a significant cause of morbidity and mortality in the tropics. Available therapies are
problematic due to toxicity, treatment duration and emerging drug resistance. Mouse models of leishmaniasis have
demonstrated that disease outcome depends critically on the balance between effector and regulatory CD4+T cell
responses, something mirrored in descriptive studies of human disease. Recombinant IL-2/diphtheria toxin fusion protein
(rIL-2/DTx), a drug that is FDA-approved for the treatment of cutaneous T cell lymphoma, has been reported to deplete
regulatory CD4+T cells.
Methodology/Principal Findings: We investigated the potential efficacy of rIL-2/DTx as adjunctive therapy for experimental
infection with Leishmania major. Treatment with rIL-2/DTx suppressed lesional regulatory T cell numbers and was associated
with significantly increased antigen-specific IFN-c production, enhanced lesion resolution and decreased parasite burden.
Combined administration of rIL-2/DTx and sodium stibogluconate had additive biological and therapeutic effects, allowing
for reduced duration or dose of sodium stibogluconate therapy.
Conclusions/Significance: These data suggest that pharmacological suppression of immune counterregulation using a
commercially available drug originally developed for cancer therapy may have practical therapeutic utility in leishmaniasis.
Rational reinvestigation of the efficacy of drugs approved for other indications in experimental models of neglected tropical
diseases has promise in providing new candidates to the drug discovery pipeline.
Citation: Divanovic S, Trompette A, Ashworth JI, Rao MB, Karp CL (2011) Therapeutic Enhancement of Protective Immunity during Experimental
Leishmaniasis. PLoS Negl Trop Dis 5(9): e1316. doi:10.1371/journal.pntd.0001316
Editor: Diane McMahon-Pratt, Yale School of Public Health, United States of America
Received July 6, 2010; Accepted August 1, 2011; Published September 6, 2011
Copyright: ? 2011 Divanovic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by US Public Health Service Grant AI057992 from the National Institutes of Health. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤ Current address: University Hospital Center of Lausanne, Rue du Bugnon, Lausanne, Switzerland
Protozoa of the genus Leishmania cause a wide spectrum of
human disease . At the severe end of the spectrum, visceral
leishmaniasis (kala azar), due to disseminated parasitism of
macrophages and dendritic cells, causes an annual mortality of
approximately 50,000, largely in India and Sudan . Kala azar
has also emerged as a significant problem in the setting of HIV/
AIDS, visceral leishmaniasis being the second most common
opportunistic tissue protozoal disease (after toxoplasmosis) in
people infected with HIV . Available therapies for kala azar,
including pentavalent antimonials, some (but not all )
amphotericin B preparations, miltefosine and paromomycin, are
problematic due to emerging drug resistance, toxicity, need for
lengthy treatment and/or the development of post-kala azar
dermal lesions [5,6,7,8,9]. There is thus a clear need for novel
therapeutic approaches to this neglected tropical disease.
Experimental mouse models of Leishmania infection have been
used extensively to interrogate the immune system as well as the
immunopathogenesis of leishmaniasis [10,11,12,13]. Inoculation
of low numbers of L. major into the dermis of C57BL/6 mice is
followed by the recruitment of antigen-specific effector CD4+and
CD8+T cells, IFN-c production at the site of infection, and
activation of the microbicidal effector functions of parasitized
macrophages, events manifested clinically by lesion development
. IL-10 production by CD4+T cells is critical to immune
counterregulation in this model. Balanced IFN-c and IL-10
responses are essential for disease resolution and the establishment
of life-long latent infection . IFN-c deficiency or neutralization
leads to systemic parasite spread [15,16]; IL-10 deficiency or
neutralization leads to sterile cure [17,18]. A similar balance
between IFN-c and IL-10 responses also appears to be a critical
determinant of the outcome of human leishmaniasis . Several
relevant IL-10-producing CD4+
described, including natural and adaptive regulatory T cells (Treg)
and Th1cells that produce IL-10 in addition to IFN-c [20,21,22].
Recent studies have emphasized the role played by the latter cells
in immune counterregulation in experimental leishmaniasis
[20,23] and human visceral leishmaniasis . That said,
monoclonal antibody-mediated depletion of CD25 (IL-2R)-
expressing cells, a technique that depletes Tregcells, has been
reported to facilitate parasite eradication in experimental leish-
maniasis, in models of primary infection and superinfection, as
well as in vaccination models [25,26,27,28].
T cell subsets have been
www.plosntds.org1September 2011 | Volume 5 | Issue 9 | e1316
Denileukin diftitox (rIL-2/diphtheria toxin [DTx]), a recombinant
fusion protein composed of the membrane-translocating and
interleukin 2 (Ala1-Thr133), is FDA-approved for the treatment of
cutaneous T cell lymphoma . Internalization of rIL-2/DTx into
cells expressing the high affinity IL-2 receptor leads to activation of
the ADP-ribosyltransferase function of DTx in the endosome.
Activated DTx is subsequently translocated into the cytosol where
it inhibits protein synthesis and induces apoptosis . rIL-2/DTx
treatment leads to a significant reduction in peripheral blood
CD4+CD25+Foxp3+Tregpopulations in humans . Furthermore,
clinical treatment of patients with rIL-2/DTx has been reported to
enhance immune responses [30,31,32]. Similarly, treatment of mice
with rIL-2/DTx has been reported to decrease splenic, bone marrow
and peripheral blood CD4+CD25+Foxp3+Treg. Such treatment
has been shown to have benefit in experimental tumor models 
and several experimental models of immune-mediated disease
Given these data, we hypothesized that rIL-2/DTx treatment
would enhance the resolution of experimental L. major infection.
Treatment with rIL-2/DTx reduced Treg/CD4+T cell ratios
during experimental L. major infection, increasing antigen-specific
IFN-c production, enhancing lesion resolution and decreasing
parasite burden. Furthermore, combined administration of rIL-2/
DTx and sodium stibogluconate had additive biological and
therapeutic effects, in both genetically resistant (C57BL/6) and
susceptible (BALB/c) mice.
Female C57BL/6 and BALB/c mice were purchased from
Jackson Laboratories. All animals were housed in a specific
pathogen-free animal facility, in high-efficiency particulate-
filtered laminar flow hoods with free access to food and water,
at Cincinnati Children’s Hospital Medical Center (CCHMC).
Animal care was provided in accordance with the procedures
outlined in the Guide for the Care and Use of Laboratory
Animals under animal study proposals approved by the CCHMC
In vivo infection model
L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were
grown at 28uC in medium 199 (Cellgro), supplemented with 20%
fetal calf serum (FCS) [Hyclone], 100 U/ml penicillin and
100 mg/ml streptomycin (Cellgro), 25 mM HEPES (Invitrogen),
2 mM L-glutamine, 0.1 mM adenine, 5 mg/ml hemin, and 2 mg/
ml d-biotin (all from Sigma), and passaged at least 3 times, but not
more than 5 times, prior to infection. Ficoll gradient purification
 was used to purify infectious phase metacyclic promastigotes
from 5 d old stationary cultures. 8 week-old mice were infected in
the dermis of the ear with 36103L. major metacyclic promastigotes
in 10 ml FBS-free media. Lesion size was quantified with vernier
calipers. All reagents used for in vivo infection were endotoxin-free
to the limits of detection of the Limulus amebocyte lysate assay (Bio-
Mice were treated intraperitoneally with rIL-2/DTx (Denileu-
kin diftitox, ONTAK; Ligand Pharmaceuticals, Inc.), intramus-
cularly with sodium stibogluconate (SSG; The Wellcome Foun-
dation, Inc., provided by the Centers for Disease Control and
Prevention), and/or an equal volume of sterile, endotoxin-free
saline (Hospira Inc.) via these same routes as a control.
To quantify lesional parasite burden, the ventral and dorsal
sheets of the infected ears were separated, deposited dermal side
down into 24-well tissue culture plates containing RPMI (Cellgro)
supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin
and 50 mg/ml liberase CI enzyme blend (Roche), and incubated
for 45 min at 37uC. Tissues were subsequently dissociated in
RPMI containing 10% FCS and 0.05% DNAse I (Sigma) using a
medimachine (BD Biosciences), according to the manufacturer’s
protocol. Tissue homogenates were filtered using a 50 mm cell
strainer (Falcon Products Inc) and serially diluted (1:2) in 96-well
flat bottom microtiter tissue culture plates containing 50 ml of
Novy-MacNeal-Nicolle (NNN) medium with 20% defibrinated
rabbit blood (Hemostat Laboratories) overlaid with 100 ml
medium 199 supplemented with 20% FCS, 100 U/ml penicillin,
100 mg/ml streptomycin, 2 mM L-glutamine, 25 mM HEPES,
0.1 mM adenine, 5 mg/ml hemin, and 2 mg/ml d-biotin. After
culture for 7 d at 28uC the number of viable parasites was
quantified by limiting dilution analysis. The parasite burden in
draining, retromaxillar lymph nodes, liver (left lobe) and spleen
was quantified by limiting dilution analysis using similar
procedures. All reagents used for cell culture and parasite titration
were endotoxin-free to the limits of detection of the Limulus
amebocyte lysate assay (Bio-Whittaker).
Flow cytometric analysis
Single cell suspensions generated from lesional sites or draining
lymph nodes, obtained as described above, were treated with
FACS fix buffer for 15 min (BD Biosciences). Cells were washed
and co-incubated with anti-FccIII/II (CD16/32; e-Bioscience)
antibody for 30 min in PBS containing 0.1% BSA and 0.01%
sodium azide. After a further wash, cells were incubated with
directly-conjugated monoclonal antibodies to TCR-b-FITC (H57-
597), CD4-PE-Cy7 (RM4-5) or CD4-APC-Alexa Fluorochrome
750 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-PE (PC61),
NK1.1-PerCp-Cy5.5 (PK136), CD49b-PE-Cy7 (DX5), F4/80-
APC (BM8), CD11b-PerCp-Cy5.5 (M1/70), Gr-1-FITC (RB6-
8C5), CD11c-Alexa Fluorochrome 700 (N418), B220-APC-Alexa
Fluorochrome 750 (RA3-6B2), and/or CD19-PE (1D3) [all
Leishmaniasis is an infectious disease that causes a large
burden of morbidity and mortality in the tropics. Caused
by protozoan parasites of the genus Leishmania that are
transmitted by sandflies, leishmaniasis causes a wide
spectrum of human disease. The severe end of the
spectrum, visceral leishmaniasis, causes an annual mortal-
ity of approximately 50,000, largely in India and Sudan.
Available therapies for leishmaniasis are problematic due
to emerging drug resistance, toxicity and/or the need for
lengthy courses of treatment. There is thus an urgent need
for novel therapeutic approaches to this neglected tropical
disease. To address this problem, the authors examined
whether a commercially available drug developed for
cancer therapy (Ontak), reported to have immunological
activity of relevance to the immunobiology of Leishmania
infection, exhibited efficacy in mouse models of leishman-
iasis. The study found therapeutic efficacy for the drug
alone in these models, as well as additive therapeutic
efficacy in combination with standard antimicrobial
therapy. Rational reinvestigation of the efficacy of already
approved drugs in experimental models of neglected
tropical diseases has promise in providing needed new
candidates to the drug discovery pipeline.
Inhibition of Counterregulation in Leishmaniasis
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antibodies were from BD Biosciences and/or e-Bioscience] for
Quantification of Foxp3 expression was done using the Foxp3-
APC (FJK-16s) staining kit (e-Bioscience) according to manufac-
turer’s instructions, combined with directly-conjugated monoclo-
nal antibodies TCR-b-FITC, CD4-APC-Alexa Fluorochrome 750
and CD25-PE (BD Biosciences and/or e-Bioscience).
Isotype control antibodies (BD Biosciences and/or e-Bioscience)
were used in each analysis. Data were collected and analyzed using
a combination of FACSCalibur flow cytometer and CellQuest
software or LSRII flow cytometer and FACSDiva software (BD
96-well, EIA/RIA flat-bottom, plates (Costar) were coated with
diphtheria toxin (1 mg/ml; Sigma) in 50 mM carbonate/bicar-
bonate buffer pH 9.6 and incubated overnight at 4uC. Plates were
washed (66) with wash buffer (Tris-Buffer Saline pH 7.2 and
0.05% Tween 20), serum samples, diluted in dilution buffer (wash
buffer supplemented with 10% SuperBlock [Pierce]), were added
and incubated for 30 min at room temperature. Plates were
washed, alkaline phosphatase-conjugated anti-mouse IgG1 anti-
body (1:1000 in dilution buffer; BD Biosciences) was added and
plates were incubated for an additional 30 min at room
temperature. After a further wash, pNPP (1 mg/ml; Calbiochem)
in TM Buffer (Tris Base supplemented with 0.3 M MgCl2,
pH 9.8) was added and optical density (405 nm) was quantified
using kinetic microplate reader (Molecular Devices).
Draining lymph node cells were plated in 96-well tissue culture
plates at 56106cells/ml and cultured for 96 h at 37uC in 5% CO2
Figure 1. Short-term treatment with rIL-2/DTx leads to transient Treg depletion. (A) Uninfected C57BL/6 mice were treated
intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 mg/kg, gray bars; 50 mg/kg, black bars) and splenic Treg
(TCRb+CD4+CD25+Foxp3+cells) were quantified by flow cytometry at the time indicated. (B) Uninfected C57BL/6 mice were treated intraperitoneally
with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 mg/kg (filled bars) and splenic Treg(TCRb+CD4+CD25+Foxp3+cells) were
quantified by flow cytometry 7 d after the final dose. (C and D) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or
rIL-2/DTx (50 mg/kg; filled bars), starting 1 week after intradermal infection in both ears with 36103metacyclic L. major promastigotes. Lesional Treg
(TCRb+CD4+CD25+Foxp3+cells) were quantified by flow cytometry (C), and IgG1antibodies to diphtheria toxin were measured by ELISA in serially
diluted serum samples (D), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/2 SE in a single experiment; n=3 (A), n=4–6 (B)
and n=5–6 (C and D). (A) ANOVA P,0.01; Tukey’s correction; *P,0.05; (B and C) Student’s t test *P,0.05, **P,0.01.
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in RPMI containing 100 U/ml penicillin, 100 mg/ml streptomy-
cin, 10% fetal calf serum, 0.1 mM b-mercaptoethanol (Invitrogen)
and soluble L. major antigens were generated as described .
Secreted IFN-c, IL-10 and IL-4 were quantified by ELISA (BD
Kinetic lesion size data was first analyzed by MANOVA, to reject
the null hypothesis of equal effects, followed by ANOVA (plus
Tukey’s multiple comparison test) or the unpaired Student’s t test, as
appropriate. In studies aimed at defining whether rIL-2/DTx
treatment allowed for a reduction in the dose or duration of standard
antimicrobial therapy, linear random effects (time) modeling was
well as to sort the therapeutic efficacy of the diverse treatment
regimens. Parasite numbers were log-transformed before analysis,
and analyzed by ANOVA (followed by Tukey’s multiple comparison
test) or the unpaired Student’s t test, or the non-parametric Kruskal-
Wallis test (followed by the Wilcoxon test), as appropriate.
Kinetics of in vivo Tregdepletion by rIL-2/DTx
Given the robust expression of the high affinity IL-2 receptor by
Foxp3-expressing Treg, it is not surprising that rIL-2/DTx treatment
has been reported to deplete Treg in humans and mice
[30,31,33,34,42,43]. To define the kinetics of rIL-2/DTx-mediated
Tregdepletion, we treated uninfected mice with a single injection of
rIL-2/DTx (or vehicle control) and quantified splenic Tregnumbers
thereafter. As shown in Figure 1A, rIL-2/DTx injection led to a
significant decrease in the percentage of splenic Tregquantified one
week after treatment (see Fig. S1 for flow cytometric gating strategy).
However this reduction was not sustained; no alterations in Treg
percentage were observed two or three weeks after administration of
a single dose (Figure 1A). A similar significant reduction in the
2/DTx (Figure 1B). However, longer treatment (8 weekly doses)
failed to result in sustained Tregdepletion (Figure 1B). During the
courseof experimental cutaneous leishmaniasis, a dynamic process of
Figure 2. Treatment with rIL-2/DTx enhances resolution of experimental L. major infection. C57BL/6 mice were infected intradermally in
both ears with 36103metacyclic L. major promastigotes. (A, B) Beginning 30 d after infection, mice were treated three times, at 5 d intervals, with
normal saline (open symbols) or rIL-2/DTx (12 mg/kg; filled symbols). (A) Lesion size; (B) Lesional parasite burden, quantified 45 d after infection. (C,
D) Beginning 7 d after infection, mice were treated at weekly intervals with normal saline (open symbols) or rIL-2/DTx (12 mg/kg; filled symbols), and
lesion size (C) and parasite burden (D) was quantified 7 d after administration of the last indicated dose. Data represent means +/2 SE of 8 mice/
group (with individual data points shown for parasite burden). (A and C) MANOVA P,0.05; (A–D) Student’s t test; *P,0.01, **P,0.005, ***P,0.001.
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Tregrecruitment to and retention in lesional sites has been observed
. Short-term administration (3 weekly doses) of rIL-2/DTx, 1
week after L. major infection, resulted in a significant reduction in
lesional, draining lymph node and splenic Tregaccumulation after 4
weeks of infection (Figure 1C and data not shown). However, similar
to findings in the spleens of uninfected mice, prolonged administra-
tion of rIL-2/DTx (7 weekly doses), 1 week after L. major infection,
failed to result in sustained lesional Tregdepletion; lesional Treg
percentages were similar in treated and mock-treated mice 8 weeks
after infection (Figure 1C). It will be noted that, with lesional healing
in these genetically resistant mice, lesional Tregnumbers decrease in
untreated mice over this time frame as well. As shown in Figure 1D,
such prolonged administration of rIL-2/DTx led to the generation of
robust titers of anti-DTx antibodies.
Short-term administration of rIL-2/DTx enhances lesion
resolution and reduces L. major parasite burden
To define the effectiveness of rIL-2/DTx administration on the
resolution of an ongoing L. major infection, mice were treated with rIL-
2/DTx, or vehicle as a control, beginning 30 d after infection. In light
of the above kinetic data, the mice were given three doses of drug or
vehicle, at 5 d intervals. As shown in Figure 2, rIL-2/DTx treatment
significantly enhanced lesion resolution (Figure 2A) and resulted in a
significant decrease in lesional parasite burden (Figure 2B).
While not especially relevant to therapy of human disease, we also
examined the effect of weekly therapy with rIL-2/DTx, beginning 1
week after infection, on experimental cutaneous leishmaniasis. This
protocol also significantly enhanced lesion resolution compared to
control therapy (Figure 2C)— something sustained from the onset of
lesionresolution inrIL-2/DTx-treated mice through the rest of the 8-
week time course of the experiment.However,while such therapyled
of rIL-2/DTx (Figure 2D), no significant enhancement (or
impairment) of host control of parasite burden was observed after 7
weekly doses of rIL-2/DTx therapy, compared with mock therapy
(Figure 2D), something perhaps predictable both from the generation
of antibodies to DTx observed with this protocol (Figure 1D), as well
as the baseline levels of host resistance observed in this model.
rIL-2/DTx and sodium stibogluconate have additive
therapeutic efficacy against experimental L. major
We next examined the therapeutic effect of co-administration of
rIL-2/DTx and pentavalent antimony (sodium stibogluconate
Figure 3. Combined rIL-2/DTx and sodium stibogluconate treatment provides additive efficacy in experimental L. major infection.
C57BL/6 mice were infected as in Figure 2. Beginning 30 d after infection, mice were treated: (1) daily for 10 d with SSG (250 mg/kg; filled triangles);
(2) three times at 5 d intervals with rIL-2/DTx (12 mg/kg; open squares); (3) three times at 5 d intervals with rIL-2/DTx (50 mg/kg; open circles); (4) daily
for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (12 mg/kg; filled squares); or (5) daily for 10 d with SSG (250 mg/kg)
plus three times at 5 d intervals with rIL-2/DTx (50 mg/kg; filled circles); or (6) with normal saline (per route and schedule for 10 d SSG plus 3 doses of
rIL-2/DTx; crossed symbols). (A) Lesion size, (B) lesional parasite burden, and (C) lesional Tregpercentage were quantified in the indicated groups,
45 d after infection. Data represent means +/2 SE of 8 mice/group in a single experiment (with individual data points shown for parasite burden);
n=8 (A and B), n=4 (C). (A) MANOVA P,0.05; (A–C) ANOVA P,0.02; Tukey’s correction; *P,0.05, **P,0.01, ***P,0.001.
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[SSG]) on the resolution of L. major infection, again, beginning
therapy 30 d after infection. Single agent therapy with either rIL-
2/DTx or SSG led to significant improvement in lesion resolution
and significant decreased parasite burden, compared to vehicle-
treated animals (Figure 3A and 3B). Further, combined therapy
with rIL-2/DTx and SSG, regardless of the dose of rIL-2/DTx
employed, resulted in significantly enhanced lesion resolution and
decreased parasite burden compared to single agent therapy
(Figure 3A and 3B). As expected, rIL-2/DTx treatment led to a
significant reduction in lesional Treg(Figure 3C). The addition of
SSG to rIL-2/DTx (or vehicle) treatment failed to alter lesional
Treg(data not shown).
Based on this, we examined the effects of the addition of rIL-2/
DTx to standard SSG therapy on a range of immune parameters
important in controlling the course of experimental infection with
L. major. Mice were treated with SSG, rIL-2/DTx and/or vehicle
controls beginning 30 days after infection. In concert with
significant effects on lesion size and lesional parasite burden
(Figure 4A and B), the addition of rIL-2/DTx to SSG led to a
significant reduction in Tregin lesions and draining lymph nodes
(Figure 4C and D). Combined therapy with rIL-2/DTx and SSG
also led to a significant increase in antigen-specific IFN-c
production by cells isolated from draining lymph nodes, compared
with control or SSG treatment alone (Figure 4E). No differences in
antigen-specific IL-10 production were observed (Figure 4F).
We subsequently examined whether the addition of rIL-2/DTx
allowed for a reduction in SSG dose or duration. Notably, as
shown in Figure 5, the added clinical benefit—reduction in lesion
size and parasite burden—afforded by adjunct therapy with rIL-
2/DTx allowed for at least a halving of the duration of SSG
treatment needed: in terms of both lesion size and parasite burden,
rIL-2/DTx+5d of SSG was more effective than the full 10d
regimen of SSG alone. Such combination therapy with rIL-2/
DTx also allowed for SSG dose sparing: rIL-2/DTx+SSG 25 mg/
kg/d for 10d had equivalent effects on lesion size and parasite
burden as SSG 250 mg/kg/d for 10d alone (Fig. 5).
Figure 4. Enhanced resolution of infection with combined therapy correlates with amplification of the effector immune response.
C57BL/6 mice were infected as in Figure 2; treatment was begun 30 d after infection. (A) Lesion size. Mice were treated: (1) daily for 10 d with SSG
(250 mg/kg; open circles); (2) daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 mg/kg; filled circles); or (3) with
normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; crossed symbols); harvested 45 d after infection. (B) Lesional parasite
burden; (C) Lesional Tregpercentage; (D) Tregpercentage in draining lymph nodes; Antigen-specific (E) IFN-c and (F) IL-10 secretion by leukocytes
isolated from lymph nodes draining lesional sites and cultured in presence of soluble Leishmania antigen (quantified by ELISA); Data represent means
+/2 SE of 6–8 animals/group in a single experiment (with individual data points shown for parasite burden); (A) MANOVA P,0.05; (A–F) statistical
analysis on data obtained 45 d after infection; ANOVA P,0.05; Tukey’s correction; *P,0.05, **P,0.001.
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Combined therapy with rIL-2/DTx and sodium
stibogluconate also exhibits additive efficacy in
genetically susceptible mice
To define the effect of rIL-2/DTx therapy on leishmaniasis in
the face of genetic susceptibility, we turned to BALB/c mice, mice
that fail to heal L. major infection. Of interest, additive therapeutic
and biological efficacy was seen even in this highly susceptible
strain. Whereas short-term therapy with either SSG or rIL-2/DTx
restrained lesion expansion, short-term combined therapy led to a
significant reduction in lesion size (Fig. 6A). Such combined
therapy also led to: (i) a significant reduction in parasite burden in
lesions, draining lymph nodes, and liver (along with a trend
towards a decrease in splenic parasite burden) (Fig. 6 B–E); (ii)
modest if significant suppression of Tregin draining lymph nodes
and spleen (Fig. 6 F and G); and (iii) significant augmentation
antigen-specific IFN-c production (in the absence of significant
effects on IL-10 and IL-4 production) by cells isolated from
draining lymph nodes (Fig. 6 H–J).
Considerable data suggest likely benefit for immunomodula-
tory approaches to therapy in leishmaniasis, a neglected tropical
infection that continues to cause a great burden of morbidity
and mortality in the tropics. Our data confirm that rIL-2/DTx
administration leads to transient depletion of TCRb+CD4+
CD25+Foxp3+Treg, demonstrating that this depletion is limited
by development of antibodies to DTx after multiple doses. Our
data further suggest potential therapeutic promise for rIL-2/
DTx in cutaneous leishmaniasis. rIL-2/DTx-mediated Treg
suppression was associated with increased antigen-specific
IFN-c production, enhanced lesion resolution and decreased
parasite burden during experimental L. major infection (in the
absence of any obvious qualitative differences in lesion
histology [data not shown]). Combined administration of
rIL-2/DTx and sodium stibogluconate had additive therapeu-
tic effects, allowing for a shortening of the needed duration or
dose of SSG therapy. It should be remarked that, whereas
Figure 5. Addition of rIL-2/DTx to antibiotic therapy allows for treatment duration reduction in experimental cutaneous
leishmaniasis. C57BL/6 mice were infected as in Figure 2. (A and B) lesion size. Beginning 30 d after infection, mice were treated as follows: (1) SSG
250 mg/kg daily for 10 d [black circles]; (2) SSG 25 mg/kg daily for 10 d [blue circles]; (3) SSG 250 mg/kg daily for 5 d [red circles]; (4) SSG 250 mg/kg
daily for 10 d+rIL-2/DTx 50 mg/kg 3 times at 5 d intervals [black squares]; (5) SSG 25 mg/kg daily for 10 d+rIL-2/DTx 50 mg/kg 3 times at 5 d intervals
[blue squares]; (6) SSG 250 mg/kg daily for 5 d+rIL-2/DTx 50 mg/kg 3 times at 5 d intervals [red squares]; (7) normal saline (per route and schedule for
10 d SSG plus 3 doses of rIL-2/DTx) [open circles]. Note, while regimens are separated into 2 panels for ease of visibility, the control (saline) group in
both panels is one and the same. (A and B) MANOVA P,0.0004; ANOVA P,0.001; Tukey’s correction; *P,0.05, **P,0.01, ***P,0.001. Linear random
effects modeling, done in a second order of analysis, rejected the null hypothesis of all treatments having equal effects (P,0.001), and sorted the
therapeutic efficacy of the regimens as follows, from best to worst: groups 4 and 6; groups 1, 3 and 5; groups 2 and 7. (C) Lesional parasite burden
quantified in the indicated groups, 45 d after infection. ANOVA P,0.001; Tukey’s correction; *P,0.05. Data represent means +/2 SE of 5–6 mice/
group in a single experiment (with individual data points shown for parasite burden).
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these data have potential therapeutic implications for human
cutaneous leishmaniasis, the implications are less clear for
visceral leishmaniasis. Treg have not been definitively been
implicated as providing a key source of immune counter-
regulation in either experimental or human visceral leishman-
iasis, settings in which it is likely that IL-10 production by
other T cells is more important [19,24,44].
The ability of rIL-2/DTx to deplete Treghas been somewhat
controversial. Studies in humans and African green monkeys that
quantified Treg by analyzing Foxp3 mRNA expression in
peripheral blood CD4+
T cells or bulk peripheral blood
mononuclear cells, or via enumeration of CD25-expressing
CD4+T cells within peripheral blood, suggested that rIL-2/DTx
administration failed to significantly deplete Treg [37,45,46].
However, direct flow cytometric quantification of Foxp3-express-
ing CD4+T cells has indicated that rIL-2/DTx treatment leads to
Tregdepletion in humans [30,31,43], something that has been
replicated in mouse models [33,34]. Our data provide insight into
the kinetics of such treatment. A single dose of rIL-2/DTx leads to
significant depletion of splenic Treg in mice, although such
depletion is reversed as early as two weeks after rIL-2/DTx
administration. Further, these data show that repetitive adminis-
tration of rIL-2/DTx leads to sustained reduction of splenic Treg
for up to four weeks of treatment in mice, something observed in
humans as well [30,45]. However, long-term repetitive adminis-
tration of rIL-2/DTx appears to be limited by the generation of
antibodies to DTx. This indicates that the efficacy of rIL-2/DTx
immunomodulation is likely to be temporally limited. This narrow
temporal window, along with the partial depletion of Tregnumbers
achieved, may actually be beneficial in limiting potential
deleterious over-activation of immune responses with sustained
Monoclonal antibody-mediated depletion of CD25-expressing
cells has been reported to facilitate parasite eradication in
experimental leishmaniasis [25,26,27,28]. Further, it should be
noted that, prior to recognition of regulatory T cells, IL-2 was
Figure 6. Combined therapy enhances resolution of experimental L. major infection in BALB/c mice. BALB/c mice were infected as in
Figure 2. (A) Lesion size. Beginning 30 d after infection, mice were treated: daily for 10 d with SSG (250 mg/kg; blue circles); 3 times at 5 d intervals
with rIL-2/DTx (50 mg/kg; red circles); daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 mg/kg; black circles); or
with normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; open circles). (B) Lesional parasite burden; (C) Parasite burden in
draining lymph nodes; (D) Parasite burden in liver (dotted line represents the limit of detection in the assay); (E) Parasite burden in spleen; (F) Splenic
Treg(TCRb+CD4+CD25+Foxp3+) percentage; (G) Draining lymph node Tregpercentage; and antigen-specific (H) IFN-c, (I) IL-10 and (J) IL-4 secretion (by
leukocytes isolated from lesional lymph nodes and cultured in presence of soluble Leishmania antigen) were quantified 45 d after infection. Data
represent means +/2 SE of 6–7 mice/group in a single experiment (with individual data points shown for parasite burden); (A) MANOVA P,0.0003;
ANOVA P,0.0001; Tukey’s correction; *P,0.05, **P,0.01, ***P,0.001. (B–E) Non-parametric ANOVA (Kruskal-Wallis test for over-all comparison of
treatment to no treatment); (B) P,0.02; (C) P,0.006; (D) not statistically significant; (E) P,0.005. Wilcoxon test (for pairwise comparisons), (B–E)
*P,0.05, **P,0.01. (F–H) ANOVA P,0.0001; Tukey’s correction; *P,0.05, **P,0.01, ***P,0.001.
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shown to be necessary for disease progression during experimental
L. major infection of susceptible murine hosts [25,47,48] (although
the effects of IL-2 manipulation on the course of experimental
infection with L. donovani appears to be more complicated,
suggesting the need for caution in extrapolating these findings to
visceral leishmaniasis [49,50]). While the effects of IL-2 on L. major
infection have remained mechanistically undefined, one of the
principal non-redundant functions of IL-2 appears to be regulation
of Treg numbers . Our finding that rIL-2/DTx-mediated
blunting of Tregnumbers is associated with increased immune-
mediated clearance of L. major is thus not unexpected. It is
acknowledged that the mechanism underlying the beneficial effects
of rIL-2/DTx therapy in experimental leishmaniasis remains
mechanistically under-defined. In particular, it may well be that
rIL-2/DTx administration results, as well, either directly or
indirectly, in biologically important changes in other cellular
subsets that modulate anti-leishmanial immune responses. Of note
in this regard, long-term depletion of CD25-expressing cells with
declizumab has been shown to increase regulatory NK cell
numbers, as well as decrease Treg numbers, in humans with
multiple sclerosis— with regulatory NK cell changes correlating
with disease suppression in this autoimmune disease [52,53].
Further, the fate of IL-10 producing Th1cells [20,23,54] following
rIL-2/DTx administration remains unclear. However, the lack of
significant differences in antigen-driven IL-10 production follow-
ing in vitro re-stimulation suggests that rIL-2/DTx administration
may not directly alter the function or the numbers of such cells.
There are many possible ways to therapeutically target Treg
numbers and/or function, including direct targeting through
CD25, blockade of IL-10, inhibition of CTLA-4 or TGF-b,
engagement of GITR, and/or activation of dendritic cells (e.g.,
through LPS or CD40) . The first two of these methods have
already shown clear efficacy in mouse models of cutaneous
leishmaniasis [14,17]. There are also, of course, theoretical reasons
for caution: therapeutic targeting of Treg has the potential for
promoting the development or expression of autoimmune disease
in susceptible hosts, and for upregulating potentially deleterious
immune responses to the infecting pathogen or to co-infecting
pathogens. Although there are similar concerns with other
immunological approaches, these considerations suggest that, for
safety, Tregtargeting should be as narrow as possible. Thus, if IL-
10 blockade and CD25+T cell targeting are both efficacious, the
latter would be preferable as IL-10 is produced by many cells other
than Treg. Similarly, while sustained targeting of Treg alone
eradicates L. major in mouse models, brief targeting of Treg, along
with antimicrobial therapy would likely be preferable. There may
also be benefit to Treg targeting in concert with therapeutic
vaccination. It should also be noted that the use of biologicals to
inhibit immunological pathways (e.g., cytokine inhibition) has, in
general, been easier and fraught with fewer side effects than the
use of biologicals to activate immunological pathways (e.g.,
cytokine therapy). Thus, inhibition of inhibitory pathways (e.g.,
targeting of CD25+) cells may be preferable to direct immune
stimulation (e.g., of dendritic cells). Together, these considerations
suggest practical therapeutic utility for direct targeting of CD25+
cells in leishmaniasis and other chronic infections in which Treg
play an important biological role in hindering host-mediated
immune clearance. More broadly, the current data suggest that
rational reinvestigation of the efficacy of drugs approved for other
indications in experimental models of neglected tropical diseases
has promise in providing needed new candidates to the drug
cells in the TCRb+CD4+gate were analyzed for CD25 and Foxp3
expression as indicated.
Gating strategy for Tregquantification. Live
Conceived and designed the experiments: SD CLK. Performed the
experiments: SD AT JIA. Analyzed the data: SD CLK MBR. Contributed
reagents/materials/analysis tools: MBR. Wrote the paper: SD CLK.
Revision and final approval of manuscript: SD AT JIA MBR CLK.
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