re-examination of the immunosuppressive mechanisms mediating non-cure of leishmania infection in...

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


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)



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



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.



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



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.



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.



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.



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.



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.



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.



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.



re-examination of the immunosuppressive mechanisms mediating non-cure of leishmania infection in...

Documents