re-examination of the immunosuppressive mechanisms mediating non-cure of leishmania infection in...
TRANSCRIPT
Re-examination of the
immunosuppressive mechanisms
mediating non-cure of Leishmaniainfection in mice
David Sacks
Charles Anderson
Authors’ address
David Sacks, Charles Anderson,
Laboratory of Parasitic Diseases, NIAID,
Bethesda, MD, USA.
Correspondence to:
David SacksLaboratory of Parasitic Diseases, NIAID
Building 4, Room 126
4 Center Drive, MSC 0425
Bethesda, MD 20892-0425
USA
Tel.: þ1 301 496 0577
Fax: þ1 301 480 3708
E-mail: [email protected]
Summary: The interleukin (IL)-4 driven, polarized T-helper 2 cell (Th2)response that controls non-healing infection with Leishmania major inBALB/c mice has long been embraced as the underlying principle withwhich to consider the pathogenesis of non-healing and systemic forms ofleishmaniasis in humans. The inability, however, to reveal a Th2 polarityassociated with non-curing clinical disease has suggested that alternativecells and cytokines are involved in susceptibility. In this review, variousmouse models of non-curing infection with L. major and other Leishmaniaspecies are re-examined in the context of the suppression mediated byIL-10 and regulatory T (Treg) cells. These activities are revealed in L. major-infected BALB/c IL-4 knockout (KO) and IL-4Ra KO mice and especiallyin non-cure resistant mice that do not default to a Th2 pathway as a resultof inherent defects in Th1 differentiation. In contrast to the extremeBALB/c susceptibility arising from an aberrant Th2 response, non-curein resistant mice arises from an imbalance in Treg cells that are activatedin the context of an ongoing Th1 response and whose primary functionmay be to suppress the immunopathology associated with persistentantiparasite responses in infected tissues.
Introduction
An effective immune response against Leishmania relies on the
differentiation of CD4þ T lymphocytes into a functionally
distinct subset, termed T-helper 1 (Th1), that induces inflam-
matory and cytotoxic responses essential for destruction of
intracellular pathogens (1, 2). The Leishmania major murine
model has been useful not only to emphasize the importance
of Th1 responses in acquired resistance to Leishmania infection,
but also it has long been and remains the prototypic model to
explore the factors controlling Th1 differentiation in vivo.
Consistent with the concept of inflammatory type 1 cytokines
as mediators of protection, genetic or acquired deficiencies
in the cytokines [interleukin (IL)-12, interferon (IFN)-g,
tumor necrosis factor-a], receptors (IL-12Ra, IFN-gR), costi-
mulatory molecules (CD40/CD40L), signaling molecules, and
Immunological Reviews 2004Vol. 201: 225–238Printed in Denmark. All rights reserved
Copyright � Blackwell Munksgaard 2004
Immunological Reviews0105-2896
225
transcription factors [signal transducer and activator of tran-
scription (STAT)-1, STAT4, T-bet] involved in the develop-
ment or function of Th1 cells will lead to susceptibility in
normally resistant mice. While immune correlates of acquired
resistance have yet to be conclusively validated in humans,
there is no compelling data to counter the prevailing view that
Th1 responses are essential and will be key to vaccine develop-
ment or immunotherapy.
Of course the exceptional appeal of the L. major murine
models is that not only can the factors controlling Th1 develop-
ment in vivo be studied in genetically resistant mice, but also
there are genetically susceptible strains, in particular BALB/c,
that make a non-protective and disease-promoting Th2
response. Countless studies in the susceptible and resistant
strains have mutually reinforced the key conclusion drawn
from each that infection outcome is determined by the balance
of Th1 and Th2 cytokines produced. This notion continues to
provide the main conceptual framework for understanding
healing versus non-healing or cutaneous versus systemic
forms of clinical disease. Yet, it is in the inability to clearly
associate a Th2 polarity with non-healing, systemic, or reacti-
vation diseases in humans that the classical L. major BALB/c
susceptibility model has faltered. As discussed in more detail
below, IFN-g producing cells or mRNA remain readily detect-
able in patients with kala-azar, post-kala-azar dermal leishma-
niasis (PKDL), or chronic forms of cutaneous leishmaniasis.
Furthermore, the opposing cytokine most commonly found in
these clinical settings is not IL-4 but IL-10. Ironically, alter-
native pathways of susceptibility are now being revealed in L.
major-infected BALB/c mice and in susceptibility models invol-
ving other Leishmania species and mouse strains that may yet
redeem the murine Leishmania infection models as being highly
relevant to clinical disease. This review attempts to reconcile
the data that have challenged the centrality of the Th2 response
in BALB/c mice in the context of regulatory T cells (Treg) that
are also activated to promote susceptibility in these mice, and
we introduce an L. major susceptibility model in normally
resistant mice that more clearly reveals the role of IL-10 and
Treg cells in non-healing infections. The relevance of IL-10
and Treg cells to concomitant immunity and clinical disease
also are discussed.
The role of early IL-4 production in Th2 polarization and
susceptibility to L. major in BALB/c mice
L. major infection in BALB/c mice leads invariably to uncon-
trolled growth of the parasite in the primary site of infection,
whether it is the footpad, the base of the tail, or the ear dermis,
and to dissemination of parasites beyond the local draining
lymph node (LN) to spleen, liver, bone marrow, and other
cutaneous sites. With some L. major strains, the parasitization of
the viscera is especially severe and the mice die, while with
others strains the systemic infection is partially contained,
perhaps due to their being refractory to growth at higher
temperature. There is consensus that the inability of BALB/c
mice to control L. major is associated with an early and sus-
tained Th2 response characterized by the expansion of IL-4-
producing CD4þ T cells. The apparent healing of L. major
infections in BALB/c mice treated around the time of infection
with anti-IL-4 monoclonal antibody (mAb) (3, 4) or in IL-4
knockout (KO) BALB/c mice (5, 6) provides solid evidence
that early IL-4 production drives the polarized Th2 response
that is responsible for suppressing Th1 development and the
high-level secretion of IFN-g required to activate infected
macrophages for parasite killing. The cellular origin of the
IL-4 produced rapidly following infection is thought to be
an oligoclonal population of Va8þVb4CD4þ T cells that
recognize the parasite antigen Leishmania homolog of receptors
for activated C-kinase (LACK), as mice deleted of Vb4þ T cells
or made tolerant to LACK by transgenic expression in the
thymus have an impaired early IL-4 response and better
control the infection (7–9). It has been suggested that LACK-
specific Vb4Va8CD4þ T cells represent a unique lineage in
BALB/c mice that are biased to produce IL-4, because their
T-cell receptor (TCR) has relatively low affinity for peptide/
major histocompatibility complex (MHC) (10).
The importance of immune deviation involving early and
sustained IL-4 production as central to L. major susceptibility is
reinforced by studies in normally resistant strains made
susceptible by genetic disruption of IFN-g or IL-12 or by
antibody neutralization of these cytokines (11–15). In each
case, susceptibility is associated with the expansion of IL-4-
producing CD4þ T cells, some of which are reactive with
LACK (16) and which if neutralized by anti-IL-4 treatment,
revert the mice to a healing phenotype. Furthermore, resistant
mice are rendered susceptible to L. major as a result of trans-
genic expression of IL-4 (17, 18) or after receiving a single
intramuscular injection of recombinant adenovirus-expressing
IL-4 (19). Thus, there appears to be little doubt that early and
sustained production of IL-4 by CD4þ T cells is a sufficient
condition to establish susceptibility to L. major. At issue is
whether the early IL-4 response by LACK-specific cells is the
underlying cause of susceptibility.
To the extent that early IL-4 production by LACK-reactive
cells controls the evolution of Th2 dominance in the murine
response to L. major, then the absence of this response in
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
226 Immunological Reviews 201/2004
resistant mice, as has been reported (13), would seem to be
the defining variable controlling the different outcomes in the
susceptible and resistant strains. This strain difference in early
LACK reactivity and IL-4 production has not, however, been a
consistent finding. In early studies, Vb4Va8 TCR usage was
found to be similar in L. major-infected BALB/c and C57BL/6
mice (20), and LACK-specific T cells also were found to
produce a burst of IL-4 in resistant B10.D2 mice (21). In
recent studies in IL-4 reporter mice, the frequency of LACK
MHC class II-tetramer binding IL-4þ cells induced early by L.
major was found to be similar in resistant and susceptible strains
(22). In fact early, albeit transient, IL-4 responses following L.
major infection in resistant mouse strains have been the more
consistent finding (15, 23–26) and might be expected, given
the influence of antigen dose on effector class commitment
(27–31), with low initial intracellular inocula biasing Th2
responses and increasing tissue parasite burdens giving way
to Th1 dominance. This bias towards early Th2 induction may
be especially strong for Leishmania, which appear to lack power-
ful ligands to activate antigen-presenting cells (APCs) to
produce IL-12.
The critical distinction between resistant and susceptible
mice in their response to L. major seems to be in their ability
or not to redirect the early IL-4 response along a normal Th1
differentiation pathway during infection. Resistant mice
appear to have an especially powerful Th1 redirecting capabil-
ity, because C3H mice that make a strong IL-4 response due to
transient treatment with IL-4 (4) or anti-IL-12 antibodies (32)
at the time of challenge ultimately revert to their normal
resistance phenotype. In contrast, BALB/c mice appear to
have an intrinsically poor Th1 differentiating capacity, because
even in the absence of IL-4 or IL-4R signaling, IFN-g responses
remain relatively low, including in those mice that are able to
control the infection (5, 33, 34).
BALB/c mice have intrinsic defects in Th1 differentiation
The requirement for IL-12/IL-12R signaling to establish and
maintain a curative Th1 response in resistant mice and the
ability of exogenous IL-12 to redirect the early IL-4 response
and to promote resistance to L. major in BALB/c mice suggest
that some failure in the IL-12 induction or response pathway
in BALB/c mice is responsible for their susceptibility. As
currently described (35), the central elements in Th1 differ-
entiation involve triggering of naı̈ve T cells through TCR and
IFN-gR, with the later signaling through STAT1 to activate T-
bet. T-bet is a master transcription factor for Th1 cytokines as
well as IL-12-receptor b2 (IL-12Rb2) expression. Although
IL-12 does not appear to activate T-bet directly, it is required
for STAT4-dependent activation of Th1-committed cells to
secrete the high levels of IFN-g needed for strong Th1 polar-
ization. Selective loss of IL-12 signaling due to down-regulated
expression of the IL-12Rb2 chain has been proposed to
explain the defective IL-12 response in BALB/c mice (36).
IL-12Rb2-chain instability in BALB/c is not necessarily IL-4
dependent (37), because it occurs in CD4þ T cells from IL-
4Ra deficient mice (33). The relevance of this intrinsic defect
has been questioned, however, by the finding that BALB/c
transgenic mice that stably express IL-12Rb2 chain and main-
tain IL-12 signaling and STAT4 activation also maintain a
non-healing phenotype (38).
It has been suggested that primary signals determine the
commitment of naı̈ve T cells even prior to the contribution of
polarizing cytokines. There is evidence that the organization of
the TCR signaling complex distinguishes Th1 and Th2 cells
and can influence cell fate decisions, with Th1 cells able to
sustain prolonged interaction with its ligand due to efficient
recruitment of TCR complex members to lipid rafts (39).
Interestingly, the colocalization of TCR and IFN-gR to lipid
rafts that occurs after TCR ligation in naı̈ve CD4þ cells from L.
major resistant mice was observed to be deficient in CD4þ cells
from BALB/c mice (L. Glimcher et al., unpublished observa-
tion). Defective formation of the signaling complex necessary
for Th1 commitment may help to explain the CD4þ T cell
intrinsic, IL-4R signaling-independent mechanism previously
reported to control the Th2 bias in activated CD4þ T cells from
BALB/c mice (40).
Additional differences in the factors controlling Th1 differ-
entiation have been described that are intrinsic not to CD4þ T
cells but to components of the innate response that help to
regulate cell fate decisions. CD11bþ dendritic cells (DCs) from
BALB/c and B10.D2 mice (also H2-d but L. major resistant)
were found to differ in their ability to polarize naı̈ve LACK-
specific and allospecific T cells CD4þ T cells in vivo and in vitro
(41). Of the parameters investigated to explain this difference,
including amounts of peptide/MHC complexes (antigen
dose), the level of expression of costimulatory molecules
(CD80/86) and the amount of pro-inflammatory cytokines
released (e.g. IL-12), the most striking difference was in the
high level of IL-1b produced by CD11bþ DCs from B10.D2
mice and the relatively low levels produced by DCs from
BALB/c mice. The CD11bþ DCs were shown to be responsible
for CD4þ T-cell priming in vivo, and they likely represent
dermal DCs, as they were CD11bþCD8–CD11cþCD40hiMHC
class IIhi, although their relationship to Langerhans cells (LCs)
was not ruled out via staining for CD205. In a closely related
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
Immunological Reviews 201/2004 227
study (42), fetal skin-derived Langerhans-like DCs from
C57BL/6 or BALB/c mice also showed intrinsic differences
in IL-1 production, in this case IL-1a, when stimulated in vitro
with amastigotes. While the role of LCs as APCs for T-cell
priming in vivo during L. major infection has yet to be conclu-
sively shown, the fact that these cells migrate to draining LNs
during L. major infection (43) and can potentially contribute to
the overall levels of IL-1 seems highly relevant, based on the
accumulating data indicating a crucial role for IL-1 in Th1
differentiation (44). Injection of IL-1 (42) or IL-1b (41)
around the time of L. major challenge enhanced protective
immunity in BALB/c mice, and IL-1 type 1 receptor-deficient
mice developed a Th2 response after L. major infection (45).
IL-1 secreted by DCs has been shown to promote Th1 differ-
entiation by activating DCs via the myeloid differentiation
factor 88 pathway to produce more IL-12p70 and to up-
regulate MHC class II and costimulatory molecules (46, 47)
and by synergizing with IL-12 for direct activation of CD4þ T
cells to produce IFN-g. In addition, the ability of IFN-g to
inhibit Th2 proliferation has been shown to be dependent on
IL-1 (48), suggesting that without sufficient amounts of IL-1,
the early IL-4 response will be resistant to inhibition by IFN-g.
It is important to note that IL-1, like other cytokines such as
IL-18, IL-23, and IL-27, seems to act as a cofactor with IL-12
to optimize IFN-g by Th1 cells; in the absence of IL-12, it is
not sufficient to instruct Th1 development. Furthermore, in
the absence of IL-1 signaling, Th1 differentiation in C57BL/6
mice was not completely ablated, and these mice still con-
trolled L. major infection (45). These results strongly suggest
that defective IL-1 production is not sufficient on its own to
account for susceptibility and that other response defects act in
concert to prevent the level of Th1 expansion required to
override the early L. major-induced IL-4 response in BALB/c
mice.
An additional host strain difference has been found in the
manner in which parasites disseminate from the site of inocu-
lation, with rapid dissemination beyond local draining LN
occurring in BALB/c mice, while parasite containment to the
footpad and draining node is observed in resistant mice (49).
The fact that the same distinctive patterns of parasite dissemi-
nation were observed in BALB/c severe combined immuno-
deficiency disease (scid) and C57Bl/6 scid mice indicates that
they are not the result of differences in the early adaptive
response (T. Kamala & P. Matzinger, unpublished observa-
tions). One result of this dissemination is that CD4þ T cells
can be found in the livers and spleens of 2-week-infected
BALB/c mice that spontaneously produce IL-4 in vitro, whereas
these cells are not found in the viscera of C57BL/6 mice (50).
The influence of the site of antigen delivery on Th-lineage
commitment has been clearly demonstrated in the L. major
model; parasites delivered intravenously or intranasally can
elicit Th2 responses and produce non-healing visceral infect-
ions in normally resistant mice (51, 52). The existence of
Th2-inducing DCs in certain tissues has been strongly sug-
gested by studies of DCs derived from liver, lung, and Peyer’s
patches (53, 54). Such populations might represent distinct
APC lineages that produce little or no IL-12 in response to
infection, or else their Th2-inducing bias might be produced
by the net effect of antigen dose, the state of DC maturation,
and tissue-specific factors, such as cytokines and chemokines
(55). The Th2 bias that is sustained in BALB/c as a result of
parasite dissemination might effectively override the Th1
priming capacity of the dermal DCs or LCs, which as noted
above is already substandard. It is interesting to speculate that
the ability of extremely low dose infection with L. major to
establish stable Th1 immunity in BALB/c mice (56), which
seems fundamentally at odds with the dosage effects on Th1/
Th2 differentiation referred to above, might be explained by
the early containment of low dose infections to the site and
local draining node, thus avoiding the Th2 priming bias that
occurs in the viscera. There is evidence to suggest that the
protection of BALB/c mice conferred by irradiated promasti-
gotes, which is critically dependent on a high dose and intra-
venous route of injection, is due not to potent priming of Th1
cells but to high dose tolerance of the L. major-specific CD4þ T
cells that would normally be activated along a Th2 develop-
mental pathway in the liver and spleen (57).
The role of other Th2 cytokines
There is thus substantial evidence to interpret BALB/c suscept-
ibility in the context of inherent defects in Th1 differentiation
pathways expressed in both T-cell and non-T-cell compart-
ments. These multiple defects are consistent with the results of
backcrossing of resistant B10.D2 mice onto susceptible BALB/
c mice, showing that at least six genetic loci, located on
chromosomes 6, 7, 10, 11, 15, and 16, contribute to resis-
tance to L. major and, furthermore, that none of these loci has a
major effect on its own (58). In serial backcrosses involving
BALB/c and another resistant strain, STS/A, at least five loci were
revealed to control susceptibility, which in most genotypes did
not correlate with Th2 responses as detected by serum IL-4 (59).
Other data have more seriously challenged the require-
ment for IL-4 in the evolution of susceptibility to L. major in
BALB/c mice. The finding that certain strains of L. major pro-
duced non-healing infections in BALB/c IL-4 KO mice (60)
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
228 Immunological Reviews 201/2004
was at first very surprising and difficult to reconcile. As IL-13
shares many biological functions with IL-4, including the use of
IL-4Ra and STAT6-dependent signaling, the finding that mice
deleted of both IL-4 and IL-13 displayed greater resistance than
either single KO strains (61), plus the greater resistance
observed in IL-4Ra versus IL-4 KO mice, strongly indicated
that IL-13 can effectively cooperate with and substitute for IL-
4 to promote Th2 differentiation and susceptibility to L. major in
BALB/c mice.
Harder to reconcile has been the observation that following
infection with certain L. major substrains, IL-4Ra KO mice or
IL-4 KO mice treated with IL-13Ra2 fusion protein to block
IL-13 biological activity remain as susceptible as wildtype or
control-treated mice (34, 62). To the extent that the suscept-
ibility in these mice is still a Th2-dependent process (an
assumption that will be challenged below), the results suggest
that under some circumstances, IL-4Ra-independent signaling
pathways exist for Th2 priming. It is now clear that IL-4R/
STAT6 signaling is not essential for priming of CD4þ T cells to
produce Th2 cytokines in vivo, because in STAT6–/– or IL-4R–/–
mice Th2 responses are decreased, but significant levels of IL-4
and other Th2-related cytokines are still present (33, 63).
Obviously, the IL-4 (or IL-13) that is produced will be unable
to instruct Th2 differentiation in the BALB/c IL-4Ra KO mice,
suggesting that the naı̈ve CD4þ T cells default along a Th2
pathway because of their inherent defects in Th1 differentia-
tion, and other Th2 cytokines are produced to help polarize
the response and promote infection. Because the BALB/c IL-
4Ra KO mice that maintained a non-healing phenotype with
certain L. major strains were able to control these infections
following transient depletion of CD4þ cells (64), an additional
Th2 cytokine was thought to be induced by infection. The
finding that double-deficient IL-4Ra� IL-10 mice and IL-4RaKO mice treated with anti-IL-10R antibody were highly resis-
tant to L. major clearly identified IL-10 as the cytokine that, at
least in the case of certain L. major strains, is necessary and
sufficient to promote susceptibility (64). Even considering
those L. major strains for which IL-4R signaling is clearly
involved in disease exacerbation, IL-10 can still be shown to
play an equally critical role, because single IL-10 KO mice and
BALB/c wild type mice treated with anti-IL-10R antibody
controlled infection better (64, 65). L. major infection in resis-
tant mice expressing an IL-10 transgene (66) or in resistant
mice treated with a recombinant adenovirus expressing IL-10
(67) produced a non-healing phenotype, suggesting that IL-10 is
as effective as IL-4 in promoting disease and Th2 differentiation.
The apparent differences in the IL-4 and IL-10 requirements
for the maintenance of BALB/c susceptibility to different L.
major strains raise the question as to how distinct susceptibility
factors can be induced by and act on such related pathogens.
On closer examination, including a more careful comparison
of parasitic load in the site of infection at multiple time points
and a more rigorous definition of ‘resistance’, it can be argued
that all L. major strains are influenced to one degree or another
by the same set of conditions. Keeping in mind that IL-4,
IL-13, and IL-10 can each have potent deactivating effects on
IFN-g-mediated killing by macrophages (68, 69), then in the
face of the inherent defects in the Th1 developmental path-
ways described above, residual production of Th2 cytokines
may be sufficient to suppress parasite killing in vivo. Thus, the
ablation of IL-4, IL-13, or IL-10 cytokines individually has in
many instances prevented or at least slowed the development
of progressive lesions but has not altered the balance in favor
of genuine resistance expressed as complete healing and a
striking reduction in tissue parasite burdens measured over
successive time points. For some L. major strains, perhaps
because they are intrinsically more resistant to immune-
mediated killing mechanisms (64), the ablation of one (IL-4
KO) or even two (IL-4Ra KO) Th2 cytokines is not sufficient
to achieve any measure of control. For these strains in parti-
cular, but arguably for all L. major strains, a global inhibition of
Th2 responses, as is accomplished in IL-4Ra KO� IL-10 KO
mice or in IL-4Ra KO mice treated with anti-IL-10R antibody,
seems necessary for the expression of a fully resistant pheno-
type, comparable to the parasite clearance and healing that is
achieved in C57BL/6 mice. This point is shown in Fig. 1,
which shows results from a large series of papers that have
been included in their analyses parasite quantitation in infected
tissues, either footpad or draining LN, determined at relatively
late time points (5–10 weeks) postchallenge with 0.1–2 million
L. major stationary phase or metacyclic promastigotes.
CD4þCD25þ Treg cells and BALB/c susceptibility
While the studies described all refine the nature of the under-
lying defect in BALB/c mice and the central role of IL-4 in the
evolution of susceptibility, they nonetheless appear to reaffirm
the relevance of the Th1/Th2 balance to the regulation of
disease outcome in vivo. In the remainder of this review, we
discuss data from published and unpublished studies that offer
an alternative immune regulatory pathway to account for
susceptibility to L. major based not on immune deviation of
CD4þ T cells toward inappropriate Th2 responses but rather
on an imbalance in homeostatic Treg cells.
The conclusion that IL-10 produced by CD4þ T cells is as
important as IL-4 in the evolution of susceptibility to L. major
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
Immunological Reviews 201/2004 229
infection in BALB/c mice raises the question of whether these
cytokines are secreted by the same Th2 cells or produced by
discreet subpopulations of CD4þ T cells that arise from differ-
ent lineages and are activated by unique priming environ-
ments. While there is little doubt based on studies of cloned
Th2 cells that IL-10 can be a component of the Th2 cytokine
profile, it is clear that IL-10 is not transcriptionally regulated in
the same manner as IL-4, IL-5, and IL-13 (i.e. by GATA-3 and
c-maf) (70), and IL-10 is not a signature Th2 cytokine, in that
it can also be expressed by other CD4þ subsets (i.e. by Treg
cells). It seems especially important to consider the possible
role of IL-10 produced by naturally occurring CD4þCD25þ
Treg cells in susceptibility to L. major, as these cells have
recently been shown to be activated during L. major infection
in resistant C57BL/6 mice to suppress the ability of L. major-
specific CD4þCD25– cells to produce a sterile cure (71).
Treg cells is the name given generally to the subsets of CD4þ
T cells, and more recently CD8þ T cells, that negatively reg-
ulate multiple immune functions. Among the different subsets
of CD4þ Treg cells that have been described, the best char-
acterized are the so-called naturally occurring CD4þCD25þ
T cells (72). In normal mice and humans, CD25þ T cells
make up 5–10% of CD4þ T cells in the blood and peripheral
lymphoid tissue. These cells develop in the thymus, where
following relatively high affinity recognition of self-peptides,
they up-regulate the transcription factor Foxp3 and the expres-
sion of CD25 (the IL-2Ra chain), which in addition to remain-
ing a constitutive marker of Treg cells in the periphery, is
essential for their survival. Treg cells play a critical role in
suppressing a number of potentially pathogenic responses in
vivo, most notably T-cell responses directed against self-
antigens. Treg cells may also suppress potentially beneficial
immune responses, such as those directed against infectious
pathogens and tumors. There is emerging evidence in experi-
mental and clinical infections, including Pneumocystis carinii
(73), Candida albicans (74), and L. major (71) in mice and
Helicobacter pylori (75) and hepatitis C in humans (76), that
Treg cells suppress effector T-cell functions and contribute,
at least in part, to pathogen persistence. In both mice and
humans, CD25þ Treg cells secrete high levels of IL-10 and
transforming growth factor (TGF)-b that are at least partially
responsible for their capacity to suppress certain pathologic or
protective immune responses in vivo. Importantly, their ability
to suppress Leishmania-specific Th1 immunity and to prevent
sterile cure in resistant mice is IL-10 dependent (71, 77).
With regard to the role of CD25þ Treg cells in L. major-
infected BALB/c mice, it is interesting that in studies predating
the description of CD25 as a constitutive marker on Treg cells,
CD45RB was used as a marker to distinguish Th2 and Th1
subsets present in infected mice (78). The removal of the
IL-4/IL-10-secreting CD4þCD45RBlow cells resulted in the
splenic transfer of immunity to L. major-infected scid mice. As
it is now known that CD4þCD25þ Treg cells bear a CD45RBlow
phenotype, it seems reasonable to reinterpret the data to
indicate that at least some of the suppressive Th2 cells were
CD25þ Treg cells. This interpretation is strengthened by the
observation that the same population of CD45RBlow cells able
to suppress L. major-specific immunity was able to protect the
BALB /c
wt
BALB/c
wt
IL-4–/
–
IL-4–/
–
IL-4
Rα–/
–
IL-4
Rα–/
–
IL-4
Rα–/
– anti-
CD4
IL-4
Rα–/
– anti-
IL-1
0R
IL-4
Rα–/
– × IL-1
0–/
–
IL-1
0–/
–
anti-
IL-4
C57BL /6
IL-4
Rα–/
– anti-
IL-1
0R
IL-4
Rα–/
– × IL-1
0–/
–
IL-1
0–/
–
anti-
CD4
anti-
IL-4
C57BL /6
108
109
107
106
105
104
103
102
101
100
108
107
106
105
104
103
102
101
100
Par
asite
num
ber
Par
asite
num
ber
A
B
Fig. 1. Multiple pathways control susceptibility to Leishmania major inBALB/c mice. (A) Parasite burdens in footpads and (B) draining lymphnodes. Data are compiled from surveyed literature (5, 6, 15, 16, 33, 34,54, 60, 62, 65, 119–127). Each data point on the graphs is the meanparasite burden from a representative data set chosen from one paper. Allmeasurements were obtained during the later phase (>5 weeks) ofinfection.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
230 Immunological Reviews 201/2004
mice against the colitis induced by the transfer of the
CD4þCD45RBhigh cells, a suppression that is now known to
involve not Th2 cells but IL-10-producing CD25þ Treg cells
(79). Another early study (80), in which biweekly adminis-
tration of anti-CD25 mAb during the first 4 weeks of infection
rendered BALB/c mice more resistant and was thought to be
due to the depletion of IL-2-dependent Th2 cells might be
reinterpreted similarly as a depletion of the naturally occurring
Treg cells. In a recent study, BALB/c IL-4Ra KO mice that
remain susceptible to L. major were able to control infection in
the footpad and ear dermis following weekly treatment with
anti-CD25 mAb (N. Noben-Trauth et al., unpublished observa-
tions). In this case, a stronger argument in favor of selective
Treg-cell depletion can be made, because Th2 development is
significantly compromised in these mice.
A final reinterpretation of past data in the context of Treg cells
is offered with regard to the well-known effects of sublethal
irradiation on reversing BALB/c susceptibility (81). While it has
been difficult to explain how sublethal irradiation might selec-
tively deplete naı̈ve CD4þ T cells that are activated by L. major to
become Th2 cells, a recent study has revealed the existence of a
cycling CD4þCD25þ Treg subset that is continuously activated
by self-antigens (82) and therefore might be especially irradia-
tion sensitive. It is also possible that the irradiation-sensitive cells
are precommitted, LACK-reactive Th2 cells that are maintained
in cycle by cross-reactive self peptides or gut antigens (83).
The relationship between Th2 and Treg responses during L.
major infection in BALB/c mice and their respective roles in
susceptibility has been further complicated by two reports
concluding that CD4þCD25þ Treg cells are activated by
L. major to suppress early Th2 responses and their removal by
a transient anti-CD25 treatment around the time of challenge
actually exacerbates infection (84, 85). Furthermore, transfer
of CD25-depleted naı̈ve syngeneic BALB/c spleen cells to scid
mice resulted in more severe lesions and enhanced IL-4
responses compared to mice transferred with total spleen
cells, again suggesting that Th2 cells are the targets of the
Treg cells in this setting and that the Th2 responses are
required for susceptibility. There is an important caveat to
this conclusion: in a follow-up paper (86), the authors noted
that if the infections were observed for a longer duration
(greater than 4 weeks), then the scid mice transferred with
naı̈ve CD25þ cells alone developed progressive lesions, while
lesions in the mice transferred with CD25– cells alone began to
regress by 3 weeks. Healing in these mice could be prevented
by transfer of CD25þ cells at 3 weeks, unless the mice were
also treated with anti-IL-10R antibody. Thus in these studies
involving subpopulations of naı̈ve CD4þCD25þ/– cells, IL-10-
producing Treg cells appear to be necessary for the transfer of
susceptibility. Many more studies will be needed to sort out
the confusion that likely stems from the different protocols
used, including transient versus sustained CD25-cell depletion,
transfers of CD25-depleted spleen as opposed to fluorescence-
activated cell sorter (FACS) sorted CD25þ/– cells, the number
and timing of cells transferred, and the duration of observation
following infection. There seems little doubt, however, that
naturally occurring Treg cells are activated during L. major
infection in BALB/c mice, and while they may inhibit Th2
responses early on, they can independently suppress host-
protective immunity and promote disease.
The role of Treg cells in non-healing L. major infections in
C57BL/6 mice
The role of Treg cells in L. major susceptibility might be more
easily studied in mouse strains that do not maintain a strong,
confounding Th2 response during infection. While the BALB/
c IL-Ra KO mice might be useful in this regard, as discussed,
their absence of IL-4R signaling does not entirely prevent Th2
development in a BALB/c background mouse with inherent
defects in Th1 development. We have recently conducted a
series of experiments in C57BL/6 mice involving an L. major
strain, NIH/Seidman (Sd), that in contrast to the majority of L.
major isolates that we and others have studied, produces non-
healing lesions in these mice (Fig. 2). It is interesting to note
that the strain was isolated from a patient who had a chronic
lesion that was refractory to chemotherapy (87). Furthermore,
the patient showed normal skin test and proliferative responses
to parasite antigens. Similarly, in the B6 mice that developed
non-healing dermal lesions, a polarized IFN-g response, with
no detectable IL-4, was measured in the CD4þ T cells from the
lesion and draining node, and the magnitude of this response
even exceeded that seen in the mice infected with a healing
strain of L. major (C. Anderson et al., unpublished observations).
Thus, Th1 response defects and Th2 immune deviation cannot
explain the failure of these mice to control infection. Fig. 3
shows the results of FACS analysis of the CD4þ cells obtained
from the L. major NIH/Sd infected ears, indicating both a high
frequency of IFN-g-producing and CD25þ cells. A role of
CD4þCD25þ Treg cells is indicated by the results of CD25
depletion experiments, which enhanced IFN-g production by
CD4þ cells in the lesion and markedly increased parasite
clearance from the site. The treatment of mice with IL-10R
antibody also has a striking effect on parasite clearance. Thus,
Treg cells and IL-10 are essential susceptibility factors in this
model and their effects seem not so much to compromise Th1
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
Immunological Reviews 201/2004 231
priming as to suppress the function of effector cells and
cytokines in the inflammatory site. As Treg cells and IL-10
are also present in healed lesions and have been shown to
prevent the complete elimination of parasites from the skin
(71), it is important to note that the number of Treg cells in
healing and non-healing lesions has not been found to be
appreciably different. Therefore, either additional factors are
induced by L. major NIH/Sd infection to promote susceptibility
or slight, difficult to measure shifts in the effector/suppressor
ratio equilibrates the site below a threshold state of activation
required for effective killing to occur.
Comparing the susceptibility phenotypes of L. major NIH/Sd-
infected BALB/c and B6 mice, the lesions in the former are more
rapidly progressive and harbor more parasites at every stage of
infection. This finding is consistent with the interpretation that
in BALB/c mice, both Treg and Th2 cells become activated to
promote disease. In the case of L. major NIH/Sd-infected IL-4 KO
and IL-4Ra KO mice, which also develop more severe non-
healing lesions than B6 mice, even if their Th2 priming path-
ways are impaired, their Treg cells will still overwhelm the
ability of an intrinsically poor Th1 response to control infection.
Susceptibility models involving other Leishmania species
The non-healing infections produced by L. major NIH/Sd in B6
mice mimics in many respects, the pattern of susceptibility
displayed by resistant mice infected with new world cutaneous
Leishmania amazonensis strains and their related species Leishmania
mexicana. In fact, the L. amazonensis studies in resistant mice
provided an early challenge to the role of IL-4 and Th2
polarization as a necessary condition for the evolution of
non-healing, disseminating forms of leishmaniasis. These
parasites produce non-healing cutaneous lesions in C3H and
C57BL/6 strains of mice (88, 89), and in some cases, the
parasites are able to disseminate to distal cutaneous and muco-
cutaneous sites (90). When infected with L amazonensis, C3H or
B6 mice do not develop a polarized Th1 response comparable
to their response induced by L. major, but neither is the
response Th2 polarized. Rather, a low level of IFN-g produc-
tion by draining LN cells is maintained during the course of
infection, and while weak and early production of IL-4 is also
seen, it is gone after 3 weeks (91, 92). The presence of IL-4 in
this infection is not necessary for susceptibility, as L. amazonen-
sis-infected IL-4 KO mice and anti-IL-4-treated mice still devel-
oped non-healing lesions with only slight or undiminished
parasite numbers in the footpad inoculation site. In addition,
the IL-4 deficiency in these mice did not enhance the IFN-gresponse or rescue the defective IL-12Rb2 expression by CD4þ
T cells from the infected mice. IL-10 was also produced by LN
cells during infection, and results from two studies involving
IL-10 KO mice indicated a role of IL-10 in promoting parasite
growth, because 1–2 log reductions were observed in the
Sd 1°
Sd 2°
Fig. 2. Non-cure Sd-infected mice areresistant to reinfection. C57BL/6 mice wereinfected in the left ear with 103 Leishmaniamajor NIH/Seidman (Sd) metacyclicpromastigotes. Fourteen weeks later, theywere re-challenged in the right ear with thesame strain and dose. The photograph wastaken 8 weeks following the re-challenge.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
232 Immunological Reviews 201/2004
deficient mice (92, 93). The levels of IFN-g, IL-12, and other
inflammatory cytokines and chemokines were also increased
in the L. amazonensis-infected IL-10 KO mice. It is important to
note that despite the reduced parasitic loads in the footpad, the
tissue pathology was only marginally improved and the lesions
did not resolve. Thus, other negative regulators of immune
functions (e.g. TGF-b) might contribute to parasite survival in
these mice, and the absence of IL-10 might produce an espe-
cially severe inflammatory response in the chronic site.
BALB/c mice are even more susceptible to L amazonensis,
developing uncontrolled lesions similar in severity to L. major.
As with L. major, IL-4 is a key susceptibility factor in L. amazo-
nensis- or L. mexicana-infected BALB/c mice because IL-4 KO
mice or mice treated with anti-IL-4 show substantially
enhanced resistance (89, 94). Furthermore, scid mice recon-
stituted with spleen cells from IL-4 KO mice but not wildtype
mice are resistant to L. mexicana (95). The Th2 polarization that
occurs in L. mexicana-infected BALB/c mice is not driven by an
early LACK-specific IL-4 response, because mice made tolerant
to LACK were still highly susceptible (96). This finding
emphasizes the point that the hyper-susceptibility of BALB/c
mice to Leishmania infection lies not in their possession of a
unique population of antigen-reactive IL-4-producing cells but
in their inherent Th1 defects that will bias Th2 priming in
response to whatever antigens are recognized early in infec-
tion. More to the theme of this review, however, is the point
that the Th2 polarization that occurs in these mice might again
obscure the contribution of other more physiologic suppres-
sive pathways. Thus, infection of BALB/c IL-10 KO mice with
L. amazonensis or L. mexicana did not appreciably alter lesion
progression, although there was an impressive 3–4 log reduc-
tion in parasite numbers in the inoculation site, and both IFN-
g and nitric oxide (NO) responses were significantly enhanced
(97). Far more impressive was the complete healing and
parasite clearance that was observed in the IL-10 KO mice
treated with anti-IL-4, which was a level of resistance that
was not achieved with anti-IL-4 treatment alone. Thus, similar
to the L. major model, complete resistance in L. amazonensis- or L.
mexicana-infected BALB/c mice requires ablation of multiple
deactivating or counter-regulatory cytokines.
Experimental visceral leishmaniasis in mice due to Leishmania
donovani, while failing to reproduce the fatal outcome and
uncontrolled parasitization of the viscera that can occur in
human kala-azar, is nonetheless associated in certain mouse
strains with a non-curative response in spleen (98). In perhaps
the earliest deviation from the precepts fostered by the L. major
BALB/c model, the non-curing infection in the B10.D2 strain
was found not to be associated with the production of Th2
cytokines, IL-4 or IL-5 (99). Subsequent studies indicated that
progressive splenic parasitization is associated with markedly
increased mRNA levels for IL-10 and TGF-b (100, 101). More
importantly, BALB/c IL-10 KO mice were found to be highly
resistant in both liver and spleen to L. donovani infection, which
correlated with increased splenic production of IFN-g and NO
(102). Furthermore, in mice with established infection, anti-
IL-10R treatment induced almost complete clearance of L.
donovani from the liver (the spleen infections were not exam-
ined) (103).
While the activation of CD4þCD25þ Treg or other Treg cell
subsets in these various Leishmania susceptibility models has yet
to be directly addressed, the identification of IL-10 as a sus-
ceptibility factor is in each case consistent with the L. major data
indicating a role for these cells in chronic infection. Whereas
104
103
102
101
100
104103102101100
104103102101100
1010
310
210
110
0
FL2
-HF
L2-H
FL3-H
FL3-H
CD4+
CD25
IFN-γ
Fig. 3. Non-healing lesions contain high numbers of Th1 and Treg
cells. Eight weeks following intradermal inoculation of C57BL/6 micewith 103 Leishmania major NIH/Seidman (Sd) metacyclic promastigotes,ears were removed and processed for lymphocyte isolation from thelesions. Cells were analyzed for CD4þCD25þ expression or re-stimulatedin vitro for measurement of intracellular interferon (IFN)-g production byCD4þ cells.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
Immunological Reviews 201/2004 233
in healing outcomes of L. major infections in B6 mice, the Treg
cells have been shown to function in equilibrium with Th1
cells to maintain latency, the more severe, non-healing forms
of leishmaniasis as described in the mouse infections involving
L. major NIH/Sd, L. amazonensis, L. mexicana, and L. donovani, may be
due to an imbalance, however slight, in the number and
activity of parasite-driven Treg cells.
Treg cells and concomitant immunity
We observed in our studies of L. major NIH/Sd infection in B6
mice that despite their inability to heal their primary site of
infection, when the animals were re-challenged in the con-
tralateral ear, they were completely protected (Fig. 2). Thus,
the Treg cells and other suppressive mechanisms that may
function within the primary inflammatory site to prevent
effective clearance by Th1 cells do not inhibit the expression
of Th1 effector activity in a naı̈ve re-challenge site. We have
argued that the activation of Treg cells during infection pro-
vides a benefit to the host by not only controlling the severity
of inflammation but also by maintaining a persistent infection,
the Treg cells will maintain an effector memory pool necessary
for resistance to reinfection. Because Treg cells may be pre-
ferentially recruited to or expanded by signals that accumulate
within inflamed tissue, they may not be able to home to naı̈ve
re-challenge sites in sufficient time to suppress the expression
of concomitant immunity.
A recent report indicates that BALB/c mice also maintain
memory or effector cells that can protect against secondary
challenge. Again, despite uncontrolled primary infection in the
ear, the mice were protected against reinfection in the other
ear (104). Because L. major-infected BALB/c mice that have
progressive disseminating lesions are thought to be a strongly
polarized Th2 mouse, a secondary infection in these mice
should be more severe if anything. The fact that protective
Th1 cells could be found in L. major-infected BALB/c mice
within the CD45RBhigh subset, as discussed above (78),
already indicates that these mice are not so polarized as initially
thought. The study by Courret et al. (104) suggests that these
cells can escape Th2 regulation and preferentially home to and
be activated within a naı̈ve re-challenge site. While this idea
may be correct, we suggest instead that at the time of re-
challenge, Th2 responses in these mice were in decline (pos-
sibly due to suppression by Treg cells), and Treg cells within
the primary site were the main cells maintaining non-cure in
these mice. This hypothesis is supported by a recent study
examining immune responses during infection of BALB/c
mice in the skin, in which the ratio of IFN-g to IL-4 began
to decline by 10 weeks, and by 15 weeks the levels had
returned to baseline, despite the fact that the primary lesion
continued to progress (105). Treg cells might be especially
dependent for their homing and activation by signals gener-
ated and accumulated within chronic inflammatory sites, and
indeed, their primary function is undoubtedly to modulate
immunopathology. While Treg cells might effectively function
to dampen the local inflammatory response and to prevent
parasite clearance from the primary site of infection, they do
not compromise the activation and recruitment of memory or
effector cells to naı̈ve re-challenge sites.
The relevance of Th2 and Treg suppressor pathways to
human disease
To the extent that the uncontrolled, disseminating infections
produced by L. major in BALB/c may be relevant to the patho-
genesis of human kala-azar, then it is important to point out
that the severity of human visceral leishmaniasis has not been
associated with increased levels of IL-4 but of IL-10, detected
in lesional tissue or in culture supernatants of peripheral blood
mononuclear cells (PBMCs) (106–109). In these studies, IL-10
levels generally declined following successful chemotherapy.
Interestingly, elevated levels of IFN-g mRNA were also found
in bone marrow, spleen, or LN biopsies of kala-azar patients,
and IFN-g-producing cells have been revealed in antigen-sti-
mulated cultures of PBMCs treated with anti-IL-10 but not
antibodies to IL-4 (110). The involvement of IL-10 in a
reactivation process is indicated by the finding that high levels
of plasma IL-10 are predictive of the development of PKDL
(111). The inflammatory cells in the PKDL lesions were
mainly CD3þ, and IL-10 and IFN-g were the most prominent
cytokines in the PKDL lesions (112, 113). With respect to
non-healing or severe lesions due to L. major in humans,
there is a report describing an association with low IFN-gand high IL-4 production by PBMCs (114). In contrast, those
studies that have analyzed the cytokine profiles in the L. major
lesion itself have reported that unfavorable evolution of loca-
lized lesions is associated with high IL-10 and IFN-g mRNA
expression (115, 116), or with in situ staining for IFN-g, IL-10,
and IL-4 (117). Furthermore, diffuse, non-healing lesions due
to Leishmania aethiopica were associated with elevated IL-10
mRNA compared to localized lesions due to the same organ-
ism, though IFN-g levels were relatively high in each case
(118). Taken together, these studies indicate that unfavorable
clinical outcomes are not related to a Th1 cell response defect
per se but to concomitant expression of IL-10. The role of Treg
cells in each of these clinical settings has yet to be defined.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
234 Immunological Reviews 201/2004
Concluding comments
L. major infection in inbred mouse strains remains a powerful
tool to study the factors controlling CD4þ T-cell subset devel-
opment in vivo. The model continues to be embraced, because
the Th1/Th2 balance that so elegantly explains resistance and
susceptibility to L. major is believed to be relevant to the clinical
outcomes associated with Leishmania and other infectious
pathogens requiring cell-mediated immunity for control. The
inability, however, to reveal a Th2 polarity associated with
many non-healing or systemic forms of leishmaniasis in
humans has questioned the relevance of the L. major BALB/c
susceptibility model to clinical disease, and it has invited the
identification of alternative cells and cytokines involved in
susceptibility. In this review, we suggest that the Th2 polar-
ization that occurs in response to L. major in BALB/c mice as a
result of their inherent defects in Th1 differentiation pathways
obscure the suppressive activities of IL-10 produced by Treg
cells. These activities can be revealed in IL-4 KO and IL-4RaKO mice to be as potent as the Th2 cells in promoting disease,
and the impairment of both suppressor pathways is required
for the development of full resistance. We introduce a model
of non-healing L. major infection in normally resistant mice, in
which the absence of a Th2 response more clearly reveals the
role of IL-10 and Treg cells in susceptibility. Furthermore, we
review the studies in mouse models involving non-healing
infections by other Leishmania species, including L. mexicana, L.
amazonensis, and L. donovani, for which a suppressive role of IL-10
and the lack of Th2 polarization have been the more consistent
findings. Taken together, it would seem that the majority of
the mouse susceptibility models argue for a suppressive path-
way that is mediated not by an aberrant Th2 response but by
an imbalance in the activities of parasite-driven Treg cells. In
contrast to susceptibility arising from immune deviation of
CD4þ T cells toward inappropriate Th2 development, the Treg
cells that are activated during Leishmania infection may have a
normal homeostatic function to control the tissue damage
associated with an ongoing Th1 response. We suggest that it
is time for these alternative suppressive pathways to more
forcefully contend with the Th1/Th2 paradigm, particularly
as there is much clinical data to support a role of IL-10 in the
regulation of concurrent Th1 immunity in non-healing and
systemic forms of leishmaniasis.
References
1. Sacks D, Noben-Trauth N. The immunology of
susceptibility and resistance to Leishmania major in
mice. Nat Rev Immunol 2002;2:845–858.
2. Gumy A, Louis J, Launois P. The murine
model of infection with Leishmania major and its
importance for the deciphering of
mechanisms underlying the differences in Th
cell differentiation in mice from different
genetic backgrounds. Int J Parasitol
2004;34:433–444.
3. Sadick MD, Heinzel FP, Holaday BJ, Pu RT,
Dawkins RS, Locksley RM. Cure of murine
leishmaniasis with anti-interleukin 4
monoclonal antibody. Evidence for a T cell-
dependent, interferon gamma-independent
mechanism. J Exp Med 1990;171:115–127.
4. Chatelain R, Varkila K, Coffman RL. IL-4 induces
a Th2 response in Leishmania major-infected mice.
J Immunol 1992;148:1182–1187.
5. Kopf M, et al. IL-4-deficient Balb/c mice
resist infection with Leishmania major. J Exp
Med 1996;184:1127–1136.
6. Mohrs M, Ledermann B, Kohler G,
Dorfmuller A, Gessner A, Brombacher F.
Differences between IL-4- and IL-4 receptor
alpha-deficient mice in chronic leishmaniasis
reveal a protective role for IL-13 receptor
signaling. J Immunol 1999;162:7302–7908.
7. Julia V, Rassoulzadegan M, Glaichenhaus N.
Resistance to Leishmania major induced by
tolerance to a single antigen. Science
1996;274:421–423.
8. Launois P, et al. IL-4 rapidly produced by V
beta 4, V alpha 8 CD4þ T cells instructs Th2
development and susceptibility to Leishmaniamajor in BALB/c mice. Immunity
1997;6:54154–54159.
9. Himmelrich H, et al. In BALB/c mice, IL-4
production during the initial phase of
infection with Leishmania major is necessary and
sufficient to instruct Th2 cell development
resulting in progressive disease. J Immunol
2000;164:4819–4825.
10. Malherbe L, et al. Selective activation and
expansion of high-affinity CD4þ T cells in
resistant mice upon infection with Leishmaniamajor. Immunity 2000;13:771–782.
11. Mattner F, et al. Genetically resistant mice
lacking interleukin-12 are susceptible to
infection with Leishmania major and mount a
polarized Th2 cell response. Eur J Immunol
1996;26:1553–1559.
12. Wang ZE, Reiner SL, Zheng S, Dalton DK,
Locksley RM. CD4þ effector cells default to
the Th2 pathway in interferon gamma-
deficient mice infected with Leishmania major. J
Exp Med 1994;179:1367–1371.
13. Launois P, Ohteki T, Swihart K, MacDonal
HR, Louis JA. In susceptible mice, Leishmaniamajor induce very rapid interleukin-4
production by CD4þ T cells which are NK
1.1–. Eur J Immunol 1995;25:3298–3307.
14. Heinzel FP, Schoenhaut DS, Rerko RM, Rosser
LE, Gately MK. Recombinant interleukin 12
cures mice infected with Leishmania major. J Exp
Med 1993;177:1505–1509.
15. Heinzel FP, Rerko RM, Ahmed F, Pearlman E.
Endogenous IL-12 is required for control of
Th2 cytokine responses capable of
exacerbating leishmaniasis in normally
resistant mice. J Immunol 1995;155:730–739.
16. Launois P, Gumy A, Himmelrich H, Locksley
RM, Rocken M, Louis JA. Rapid IL-4
production by Leishmania homolog of
mammalian RACK1-reactive CD4(þ) T cells
in resistant mice treated once with anti-IL-12
or -IFN-gamma antibodies at the onset of
infection with Leishmania major instructs Th2
cell development, resulting in nonhealing
lesions. J Immunol 2002;168:4628–4635.
17. Erb KJ, Blank C, Moll H. Susceptibility to
Leishmania major in IL-4 transgenic mice is not
correlated with the lack of a Th1 immune
response. Immunol Cell Biol 1996;74:239–244.
18. Leal LM, Moss DW, Kuhn R, Muller W, Liew
FY. Interleukin-4 transgenic mice of resistant
background are susceptible to Leishmania majorinfection. Eur J Immunol 1993;23:566–569.
19. Gabaglia CR, et al. A single intramuscular
injection with an adenovirus-expressing IL-12
protects BALB/c mice against Leishmania majorinfection, while treatment with an IL-4-
expressing vector increases disease susceptibility
in B10.D2 mice. J Immunol 1999;162:753–760.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
Immunological Reviews 201/2004 235
20. Reiner SL, Wang ZE, Hatam F, Scott P,
Locksley RM. TH1 and TH2 cell antigen
receptors in experimental leishmaniasis.
Science 1993;259:1457–1460.
21. Julia V, Glaichenhaus N. CD4(þ) T cells
which react to the Leishmania major LACK
antigen rapidly secrete interleukin-4 and are
detrimental to the host in resistant B10.D2
mice. Infect Immun 1999;67:3641–3644.
22. Stetson DB, Mohrs M, Mattlet-Designe V,
Teyton L, Locksley RM. Rapid expansion and
IL-4 expression by Leishmania-specific naive
helper T cells in vivo. Immunity
2002;17:191–200.
23. Morris L, Troutt AB, Handman E, Kelso A.
Changes in the precursor frequencies of IL-4
and IFN-gamma secreting CD4þ cells
correlate with resolution of lesions in murine
cutaneous leishmaniasis. J Immunol
1992;149:2715–2721.
24. Belkaid Y, Mendez S, Lira R, Kadambi N,
Milon G, Sacks D. A natural model of
Leishmania major infection reveals a prolonged
‘silent’ phase of parasite amplification in the
skin before the onset of lesion formation and
immunity. J Immunol 2000;165:969–977.
25. Reiner SL, Zheng S, Wang ZE, Stowring L,
Locksley RM. Leishmania promastigotes
evade interleukin 12 (IL-12) induction by
macrophages and stimulate a broad range of
cytokines from CD4þ T cells during initiation
of infection. J Exp Med 1994;179:447–456.
26. Scott P, Eaton A, Gause WC, di Zhou X,
Hondowicz B. Early IL-4 production does not
predict susceptibility to Leishmania major. Exp
Parasitol 1996;84:178–187.
27. Constant S, Pfeiffer C, Woodard A, Pasqualini
T, Bottomly K. Extent of T cell receptor
ligation can determine the functional
differentiation of naive CD4þ T cells. J Exp
Med 1995;182:1591–1596.
28. Tao X, Constant S, Jorritsma P, Bottomly K.
Strength of TCR signal determines the
costimulatory requirements for Th1 and Th2
CD4þ T cell differentiation. J Immunol
1997;159:5956–5963.
29. Badou A, et al. Weak TCR stimulation induces
a calcium signal that triggers IL-4 synthesis,
stronger TCR stimulation induces MAP
kinases that control IFN-gamma production.
Eur J Immunol 2001;31:2487–2496.
30. O’Garra A. Cytokines induce the development
of functionally heterogeneous T helper cell
subsets. Immunity 1998;8:275–283.
31. Croft M, Rogers PR. High antigen density and
IL-2 are required for generation of CD4
effectors secreting Th1 rather than Th0
cytokines. J Immunol 1999;163:1205–1213.
32. Hondowicz BD, Scharton-Kersten TM, Jones
DE, Scott P. Leishmania major-infected C3H mice
treated with anti-IL-12 mAb develop but do
not maintain a Th2 response. J Immunol
1997;159:5024–5031.
33. Mohrs M, Holscher C, Brombacher F.
Interleukin-4 receptor alpha-deficient BALB/c
mice show an unimpaired T helper 2
polarization in response to Leishmania majorinfection. Infect Immun 2000;68:1773–1780.
34. Noben-Trauth N, Paul WE, Sacks DL.
IL-4- and IL-4 receptor-deficient BALB/c
mice reveal differences in susceptibility to
Leishmania major parasite substrains. J Immunol
1999;162:6132–6140.
35. Szabo SJ, Sullivan BM, Peng SL, Glimcher LH.
Molecular mechanisms regulating Th1
immune responses. Annu Rev Immunol
2003;21:713–758.
36. Hondowicz BD, Park AY, Elloso MM, Scott P.
Maintenance of IL-12-responsive CD4þ
T cells during a Th2 response in Leishmaniamajor-infected mice. Eur J Immunol
2000;30:2007–2014.
37. Himmelrich H, Parra-Lopez C, Tacchini-
Cottier F, Louis JA, Launois P. The IL-4 rapidly
produced in BALB/c mice after infection with
Leishmania major down-regulates IL-12 receptor
beta 2-chain expression on CD4þ T cells
resulting in a state of unresponsiveness to
IL-12. J Immunol 1998;161:6156–6163.
38. Nishikomori R, Gurunathan S, Nishikomori
K, Strober W. BALB/c mice bearing a
transgenic IL-12 receptor beta 2 gene exhibit
a nonhealing phenotype to Leishmania majorinfection despite intact IL-12 signaling.
J Immunol 2001;166:6776–6783.
39. Balamuth F, Leitenberg D, Unternaehrer J,
Mellman I, Bottomly K. Distinct patterns of
membrane microdomain partitioning in Th1
and Th2 cells. Immunity 2001;15:729–738.
40. Bix M, Wang ZE, Thiel B, Schork NJ, Locksley
RM. Genetic regulation of commitment to
interleukin 4 production by a CD4(þ)
T cell-intrinsic mechanism. J Exp Med
1998;188:2289–2299.
41. Filippi C, Hughes S, Cazareth J, Julia N,
Glaichenhaus N, Ugolini S. CD4þ T cell
polarization in mice is modulated by strain-
specific major histocompatibility complex-
independent differences within dendritic
cells. J Exp Med 2003;198:201–209.
42. Von Stebut E, et al. Interleukin 1 alpha
promotes Th1 differentiation and inhibits
disease progression in Leishmania major-susceptible BALB/c mice. J Exp Med
2003;198:191–199.
43. Moll H. The role of dendritic cells at the early
stages of Leishmania infection. Adv Exp Med
Biol 2000;479:163–173.
44. Iwasaki A. The importance of CD11bþdendritic cells in CD4þ T cell activation in
vivo: with help from interleukin 1. J Exp Med
2003;198:185–190.
45. Satoskar AR, Nishizaki K, Abe M, Harn DA Jr.
Enhanced Th2-like responses in IL-1 type 1
receptor-deficient mice. J Immunol
1999;163:6712–6717.
46. Shornick LP, Bisarya AK, Chaplin DD. IL-
1beta is essential for Langerhans cell
activation and antigen delivery to the lymph
nodes during contact sensitization: evidence
for a dermal source of IL-1beta. Cell Immunol
2001;211:105–112.
47. Eriksson U, et al. Activation of dendritic cells
through the interleukin 1 receptor 1 is critical
for the induction of autoimmune
myocarditis. J Exp Med 2003;197:323–331.
48. Oriss TB, McCarthy SA, Morel BF, Campana
MA, Morel PA. Crossregulation between T
helper cell (Th) 1 and Th2: inhibition of Th2
proliferation by IFN-gamma involves
interference with IL-1. J Immunol
1997;158:3666–3672.
49. Laskay T, Diefenbach A, Rollinghoff M, Solbach
W. Early parasite containment is decisive for
resistance to Leishmania major infection. Eur J
Immunol 1995;25:2220–2227.
50. Yamashita T, et al. CD4þ and/or
gammadeltaþ T cells in the liver
spontaneously produce IL-4 in vitro during
the early phase of Leishmania major infection in
susceptible BALB/c mice. Acta Trop
1999;73:109–119.
51. Nabors GS, Nolan T, Croop W, Li J, Farrell JP.
The influence of the site of parasite
inoculation on the development of Th1 and
Th2 type immune responses in (BALB/c x
C57BL/6), F1 mice infected with Leishmaniamajor. Parasite Immunol 1995;17:569–579.
52. Constant SL, Lee KS, Bottomly K. Site of
antigen delivery can influence T cell priming:
pulmonary environment promotes
preferential Th2-type differentiation. Eur J
Immunol 2000;30:840–847.
53. Khanna A, Morelli AE, Zhong C, Takayama T,
Lu L, Thomson AW. Effects of liver-derived
dendritic cell progenitors on Th1- and Th2-
like cytokine responses in vitro and in vivo.
J Immunol 2000;164:1346–1354.
54. Iwasaki A, Kelsall BL. Freshly isolated Peyer’s
patch, but not spleen, dendritic cells produce
interleukin 10 and induce the differentiation
of T helper type 2 cells. J Exp Med
1999;190:229–239.
55. Boonstra A, et al. Flexibility of mouse classical
and plasmacytoid-derived dendritic cells in
directing T helper type 1 and 2 cell
development: dependency on antigen dose
and differential toll-like receptor ligation.
J Exp Med 2003;197:101–109.
56. Bretscher PA, Wei G, Menon JN, Bielefeldt-
Ohmann H. Establishment of stable, cell-
mediated immunity that makes ‘susceptible’
mice resistant to Leishmania major. Science
1992;257:539–542.
57. Aebischer T, Morris L, Handman E.
Intravenous injection of irradiated Leishmaniamajor into susceptible BALB/c mice:
immunization or protective tolerance. Int
Immunol 1994;6:1535–1543.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
236 Immunological Reviews 201/2004
58. Beebe AM, Mauze S, Schork NJ, Coffman RL.
Serial backcross mapping of multiple loci
associated with resistance to Leishmania major in
mice. Immunity 1997;6:551–557.
59. Lipoldova M, et al. Susceptibility to Leishmaniamajor infection in mice: multiple loci and
heterogeneity of immunopathological
phenotypes. Genes Immun 2000;1:200–206.
60. Noben-Trauth N, Kropf P, Muller I.
Susceptibility to Leishmania major infection in
interleukin-4-deficient mice. Science
1996;271:987–990.
61. Matthews DJ, Emson CL, McKenzie GJ, Jolin
HE, Blackwell JM, McKenzie AN. IL-13 is a
susceptibility factor for Leishmania majorinfection. J Immunol 2000;164:1458–1462.
62. Kropf P, et al. Expression of Th2 cytokines
and the stable Th2 marker ST2L in the
absence of IL-4 during Leishmania majorinfection. Eur J Immunol
1999;29:3621–3628.
63. Jankovic D, Kullberg MC, Noben-Trauth N,
Caspar P, Paul WE, Sher A. Single cell analysis
reveals that IL-4 receptor/Stat6 signaling is
not required for the in vivo or in vitro
development of CD4þ lymphocytes with a
Th2 cytokine profile. J Immunol
2000;164:3047–3055.
64. Noben-Trauth N, Lira R, Nagase H, Paul WE,
Sacks DL. The relative contribution of IL-4
receptor signaling and IL-10 to susceptibility
to Leishmania major. J Immunol
2003;170:5152–5158.
65. Kane MM, Mosser DM. The role of IL-10 in
promoting disease progression in leishmaniasis.
J Immunol 2001;166:1141–1147.
66. Groux H, et al. A transgenic model to analyze
the immunoregulatory role of IL-10 secreted
by antigen-presenting cells. J Immunol
1999;162:1723–1729.
67. Viana da Costa A, Huerre M, Delacre M,
Auriault C, Correia Costa JM, Verwaerde C.
IL-10 leads to a higher parasite persistence in
a resistant mouse model of Leishmania majorinfection. Parasitol Int 2002;51:367–379.
68. Bogdan C, Nathan C. Modulation of
macrophage function by transforming
growth factor beta, interleukin-4, and
interleukin-10. Ann N Y Acad Sci
1993;685:713–739.
69. Bogdan C, Thuring H, Dlaska M, Rollinghoff
M, Weiss G. Mechanism of suppression of
macrophage nitric oxide release by IL-13:
influence of the macrophage population.
J Immunol 1997;159:4506–4513.
70. Glimcher LH, Murphy KM. Lineage
commitment in the immune system: the
T helper lymphocyte grows up. Genes Dev
2000;14:1693–1711.
71. Belkaid Y, Piccirillo CA, Mendez S, Shevach
EM, Sacks DL. CD4þCD25þ regulatory T cells
control Leishmania major persistence and
immunity. Nature 2002;420:502–507.
72. Rudensky AY, Gavin MA. Homeostasis and
anergy of CD4(þ) CD25(þ) suppressor T
cells in vivo. Nat Immunol 2002;3:109–110.
73. Hori S, Carvalho TL, Demengeot J.
CD25þCD4þ regulatory T cells suppress
CD4þ T cell-mediated pulmonary
hyperinflammation driven by Pneumocystiscarinii in immunodeficient mice. Eur J
Immunol 2002;32:1282–1291.
74. Montagnoli C, et al. B7/CD28-dependent
CD4þCD25þ regulatory T cells are essential
components of the memory-protective
immunity to Candida albicans. J Immunol
2002;169:6298–6308.
75. Lundgren A, Suri-Payer E, Enarsson K,
Svennerholm AM, Lundin BS. Helicobacterpylori-specific CD4þ CD25 high regulatory
T cells suppress memory T-cell responses
to H. pylori in infected individuals. Infect
Immunity 2003;71:1755–1762.
76. Clouston AD, et al. CD4 T helper type 1 and
regulatory T cells induced against the same
epitopes on the core protein in hepatitis C
virus-infected persons. Gut 2002;51:89–94.
77. Belkaid Y, et al. The role of interleukin (IL)-10
in the persistence of Leishmania major in the skin
after healing and the therapeutic potential of
anti-IL-10 receptor antibody for sterile cure.
J Exp Med 2001;194:1497–1506.
78. Powrie F, Correa-Oliveira R, Mauze S,
Coffman RL. Regulatory interactions between
CD45RBhigh and CD45RBlow CD4þ T cells
are important for the balance between
protective and pathogenic cell-mediated
immunity. J Exp Med 1994;179:589–600.
79. Singh B, et al. Control of intestinal
inflammation by regulatory T cells. Immunol
Rev 2001;182:190–200.
80. Heinzel FP, Rerko RM, Hatam F, Locksley
RM. IL-2 is necessary for the progression of
leishmaniasis in susceptible murine hosts.
J Immunol 1993;150:3924–3931.
81. Howard JG, Hale C, Liew FY. Immunological
regulation of experimental cutaneous
leishmaniasis. IV. Prophylactic effect of sublethal
irradiation as a result of abrogation of suppressor
T cell generation in mice genetically susceptible to
Leishmania tropica. J Exp Med 1981;153:557–568.
82. Fisson S, et al. Continuous activation of
autoreactive CD4þ CD25þ regulatory T cells in
the steady state. J Exp Med 2003;198:737–746.
83. Filippi C, et al. Priming by microbial antigens
from the intestinal flora determines the
ability of CD4þ T cells to rapidly secrete IL-4
in BALB/c mice infected with Leishmania major.Immunity 2000;13:771–782.
84. Aseffa A, Gumy A, Launois P, MacDonald HR,
Louis JA, Tacchini-Cottier F. The early IL-4
response to Leishmania major and the resulting
Th2 cell maturation steering progressive
disease in BALB/c mice are subject to the
control of regulatory CD4þCD25þ T cells.
J Immunol 2002;169:3232–3241.
85. Xu D, et al. CD4þCD25þ regulatory T cells
suppress differentiation and functions of Th1
and Th2 cells, Leishmania major infection, and
colitis in mice. J Immunol 2003;170:394–399.
86. Liu H, Hu B, Xu D, Liew FY. CD4þCD25þ
regulatory T cells cure murine colitis: the role
of IL-10, TGF-beta, and CTLA4. J Immunol
2003;171:5012–5017.
87. Neva FA. Cutaneous leishmaniasis – a case
with persistent organisms after treatment in
presence of normal immune response. Am J
Trop Med Hyg 1979;28:472–479.
88. Roberts M, Alexander J, Blackwell JM. Genetic
analysis of Leishmania mexicana infection in
mice: single gene (Scl-2) controlled
predisposition to cutaneous lesion
development. J Immunogenet 1990;17:
89–100.
89. Afonso L, Scott P. Immune responses
associated with susceptibility of C57BL/10
mice to Leishmania amazonensis. Infect Immun
1993;61:2952–2959.
90. Barral A, Petersen EA, Sacks DL, Neva FA. Late
metastatic Leishmaniasis in the mouse. A
model for mucocutaneous disease. Am J Trop
Med Hyg 1983;32:277–285.
91. Jones DE, Buxbaum LU, Scott P. IL-4-
independent inhibition of IL-12
responsiveness during Leishmania amazonensisinfection. J Immunol 2000;165:364–372.
92. Ji J, Sun J, Soong L. Impaired expression of
inflammatory cytokies and chemokines at
early stages of infection with Leishmaniaamazonensis. Infect Immun 2003;71:
4278–4288.
93. Jones DE, Ackermann M, Wille U, Hunter CA,
Scott P. Early enhanced Th1 response after
Leishmania amazonensis infection of C57BL/6
interleukin-10-deficient mice does not lead
to resolution of infection. Infect Immun
2002;70:2151–2158.
94. Satoskar A, Bluethmann H, Alexander J.
Disruption of the murine interleukin-4 gene
inhibits disease progression during Leishmaniamexicana infection but does not increase
control of Leishmania donovani infection. Infect
Immun 1995;63:4894–4899.
95. Satoskar A, et al. SCID mice reconstituted
with IL-4-deficient lymphocytes, but not
immunocompetent lymphocytes, are
resistant to cutaneous leishmaniasis.
J Immunol 1997;159:5005–5013.
96. Torrentera FA, Glaichenhaus N, Laman J,
Carlier Y. T-cell responses to
immunodominant LACK antigen do not play
a critical role in determining susceptibility of
BALB/c mice to Leishmania mexicana. Infect
Immun 2001;69:617–621.
97. Padigel UM, Alexander J, Farrell J. The role of
interleukin-10 in susceptibility of BALB/c
mice to infection with Leishmania mexicana and
Leishmania amazonensis. J Immunol
2003;171:3705–3710.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
Immunological Reviews 201/2004 237
98. Blackwell J, Ulczak OM. Immunoregulation
of genetically controlled acquired resistance
to Leishmania donovani infection in mice:
demonstration and characterization of
suppressor T cells in noncure mice. Infect
Immun 1984;44:97–102.
99. Kaye PM, Curry AJ, Blackwell JM.
Differential production of Th1- and Th2-
derived cytokines does not determine the
genetically controlled or vaccine-induced
rate of cure in murine visceral leishmaniasis.
J Immunol 1991;146:2763–2770.
100. Melby PC, Yang Y, Cheng J, Zhao W.
Regional differences in the cellular immune
response to experimental cutaneous or
visceral infection with Leishmania donovani.Infect Immun 1998;66:18–27.
101. Melby PC, Tabares A, Restrepo BI, Cardona
AE, McGuff HS, Teale JM. Leishmania donovani:evolution and architecture of the splenic
cellular immune response related to control
of infection. Exp Parasitol 2001;99:17–25.
102. Murphy ML, Wille U, Villegas EN, Hunter
CA, Farrell JP. IL-10 mediates susceptibility
to Leishmania donovani infection. Eur J
Immunol 2001;31:2848–2856.
103. Murray H, et al. Interleukin-10 in
experimental visceral leishmaniasis and IL-
10 receptor blockade as immunotherapy.
Infect Immun 2002;70:6284–6293.
104. Courret N, Lang T, Milon G, Antoine JC.
Intradermal inoculations of low doses of
Leishmania major and Leishmania amazonensismetacyclic promastigotes induce different
immunoparasitic processes and status of
protection in BALB/c mice. Int J Parasitol
2003;33:1373–1383.
105. Baldwin TM, Elso C, Curtis J, Buckingham L,
Handman E. The site of Leishmania majorinfection determines disease severity and
immune responses. Infect Immun
2003;71:6830–6834.
106. Karp CL, et al. In vivo cytokine profiles in
patients with kala-azar. Marked elevation of
both interleukin-10 and interferon-gamma.
J Clin Invest 1993;91:1644–1648.
107. Ghalib HW, et al. Interleukin 10 production
correlates with pathology in human
Leishmania donovani infections. J Clin Invest
1993;92:324–329.
108. Ribeiro-de-Jesus A, Almeida RP, Lessa H,
Bacellar O, Carvalho EM. Cytokine profile
and pathology in human leishmaniasis. Braz
J Med Biol Res 1998;31:143–148.
109. Kenney RT, Sacks DL, Gam AA, Murray HW,
Sundar S. Splenic cytokine responses in
Indian kala-azar before and after treatment. J
Infect Dis 1998;177:815–818.
110. Holaday BJ, et al. Potential role for
interleukin-10 in the immunosuppression
associated with kala azar. J Clin Invest
1993;92:2626–2632.
111. Gasim S, et al. High levels of plasma IL-10
and expression of IL-10 by keratinocytes
during visceral leishmaniasis predict
subsequent development of post-kala-azar
dermal leishmaniasis. Clin Exp Immunol
1998;111:64–69.
112. Ismail A, et al. Immunopathology of post
kala-azar dermal leishmaniasis (PKDL): T-
cell phenotypes and cytokine profile. J
Pathol 1999;189:615–622.
113. Gasim S, Elhassan AM, Kharazmi A, Khalil
EA, Ismail A, Theander TG. The
development of post-kala-azar dermal
leishmaniasis (PKDL) is associated with
acquisition of Leishmania reactivity by
peripheral blood mononuclear cells
(PBMC). Clin Exp Immunol 2000;119:523–
529.
114. Ajdary S, Alimohammadian MH, Eslami MB,
Kemp K, Kharazmi A. Comparison of the
immune profile of nonhealing cutaneous
Leishmaniasis patients with those with
active lesions and those who have recovered
from infection. Infect Immun
2000;68:1760–1764.
115. Melby PC, Andrade-Narvaez FJ, Darnell BJ,
Valencia-Pacheco G, Tryon VV, Palomo-
Cetina A. Increased expression of
proinflammatory cytokines in chronic
lesions of human cutaneous leishmaniasis.
Infect Immun 1994;62:837–842.
116. Louzir H, et al. Immunologic determinants
of disease evolution in localized cutaneous
leishmaniasis due to Leishmania major. J Infect
Dis 1998;177:1687–1695.
117. Gaafar A, Veress B, Permin H, Kharazmi A,
Theander TG, el Hassan AM.
Characterization of the local and systemic
immune responses in patients with
cutaneous leishmaniasis due to Leishmaniamajor. Clin Immunol 1999;91:314–320.
118. Akuffo H, Maasho K, Blostedt M, Hojeberg
B, Britton S, Bakhiet M. Leishmania aethiopicaderived from diffuse leishmaniasis patients
preferentially induce mRNA for interleukin-
10 while those from localized leishmaniasis
patients induce interferon-gamma. J Infect
Dis 1997;175:737–741.
119. Nabors GS, Farrell JP. Depletion of
Interleukin-4 in BALB/c mice with
established Leishmania major infections
increases the efficacy of antimony therapy
and promotes Th1-like responses. Infect
Immun 1994;62:5498–5504.
120. Kropf P, Etges R, Schopf L, Chung C, Sypek
J, Muller I. Characterization of T cell-
mediated responses in nonhealing and
healing Leishmania major infections in the
absence of endogenous IL-4. J Immunol
1997;159:3434–3443.
121. Wilhelm P, et al. Rapidly fatal leishmaniasis
in resistant C57BL/6 mice lacking TNF. J
Immunol 2001;166:4012–4019.
122. Kamanaka M, et al. Protective role of CD40
in Leishmania major infection at two distinct
phases of cell-mediated immunity.
Immunity 1996;4:275–281.
123. Monteforte GM, Takeda K, Rodriguez-Sosa
M, Akira S, David JR, Satoskar AR.
Genetically resistant mice lacking IL-18 gene
develop Th1 response and control cutaneous
Leishmania major infection. J Immunol
2000;164:5890–5893.
124. Padigel UM, Perrin PJ, Farrell JP. The
development of a Th1-type response and
resistance to Leishmania major infection in the
absence of CD40-CD40L costimulation. J
Immunol 2001;167:5874–5879.
125. Chakour R, et al. Both the Fas ligand and
inducible nitric oxide synthase are needed
for control of parasite replication within
lesions in mice infected with Leishmania majorwhereas the contribution of tumor necrosis
factor is minimal. Infect Immun
2003;71:5287–5295.
126. Mattner F, Di Padova K, Alber G.
Interleukin-12 is indispensable for
protective immunity against Leishmania major.Infect Immun 1997;65:4378–4383.
127. Satoskar A, Okano M, David JR. Gammadelta
T cells are not essential for control of
cutaneous Leishmania major infection in
genetically resistant C57BL/6 mice. J Infect
Dis 1997;176:1649–1652.
Sacks & Anderson � Immunosuppressive mechanisms in leishmaniasis
238 Immunological Reviews 201/2004