Protective response to Leishmania major in BALB/c mice requires antigen processing in the absence of DM.
ABSTRACT Protection from the parasite Leishmania major is mediated by CD4 T cells. BALB/c mice are susceptible to L. major and show a nonprotective immunodominant CD4 T cell response to Leishmania homolog of activated receptor for c-kinase (LACK) 158-173. Host genes that underlie BALB/c susceptibility to L. major infections are poorly defined. DM, a nonclassical MHC class II molecule, due to its peptide editing properties has been shown to 1) edit the repertoire of peptides displayed by APC, and 2) focus the display of epitopes by APC to the immunodominant ones. We tested the hypothesis that deficiency of DM, by causing presentation of a different array of epitopes by infected APC than that presented by DM-sufficient APC, may change the course of L. major infection in the susceptible BALB/c mice. We show herein that unlike their susceptible wild-type counterparts, BALB/c mice deficient in DM are protected from infections with L. major. Furthermore, DM-deficient mice fail to display the immunodominant LACK 158-173 on infected APC. In its place, infected DM(-/-) hosts show elicitation of CD4 T cells specific for newer epitopes not presented by wild-type L. major-infected APC. Protection of BALB/c DM(-/-) mice is dependent on IFN-gamma. DM is thus a host susceptibility gene in BALB/c mice, and Ag processing in the absence of DM results in elicitation of a protective T cell response against L. major infections. This report suggests a novel mechanism to trigger host resistance against pathogens.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: Control of parasitic protozoan infections requires the generation of efficient innate and adaptive immune responses, and in most cases both CD8 and CD4 T cells are necessary for host survival. Since intracellular protozoa remodel the vacuolar compartments in which they reside, it is not obvious how their antigens enter the MHC class I and class II pathways. Studies using genetically engineered parasites have shown that host cell targeting, intracellular compartmentalization, subcellular localization of antigen within the parasite, and mechanism of invasion are important factors determining the presentation pathway utilized. The recent identification of endogenous parasite-derived CD8 T cell epitopes have helped confirm these concepts as well as provided new information on the processing pathways and the impact of parasite-stage specific antigen expression on the repertoire of responding T cells stimulated by infection. Elucidating the mechanisms governing antigen processing and presentation of intracellular protozoa may provide important insights needed for the rational design of effective vaccines.Current opinion in immunology 02/2010; 22(1):118-23. · 10.88 Impact Factor
Protective Response to Leishmania major in BALB/c Mice
Requires Antigen Processing in the Absence of DM1
Tirumalai Kamala* and Navreet K. Nanda2*†
Protection from the parasite Leishmania major is mediated by CD4 T cells. BALB/c mice are susceptible to L. major and show a
nonprotective immunodominant CD4 T cell response to Leishmania homolog of activated receptor for c-kinase (LACK) 158–173.
Host genes that underlie BALB/c susceptibility to L. major infections are poorly defined. DM, a nonclassical MHC class II
molecule, due to its peptide editing properties has been shown to 1) edit the repertoire of peptides displayed by APC, and 2) focus
the display of epitopes by APC to the immunodominant ones. We tested the hypothesis that deficiency of DM, by causing
presentation of a different array of epitopes by infected APC than that presented by DM-sufficient APC, may change the course
of L. major infection in the susceptible BALB/c mice. We show herein that unlike their susceptible wild-type counterparts, BALB/c
mice deficient in DM are protected from infections with L. major. Furthermore, DM-deficient mice fail to display the immuno-
dominant LACK 158–173 on infected APC. In its place, infected DM?/?hosts show elicitation of CD4 T cells specific for newer
epitopes not presented by wild-type L. major-infected APC. Protection of BALB/c DM?/?mice is dependent on IFN-?. DM is thus
a host susceptibility gene in BALB/c mice, and Ag processing in the absence of DM results in elicitation of a protective T cell
response against L. major infections. This report suggests a novel mechanism to trigger host resistance against pathogens. The
Journal of Immunology, 2009, 182: 4882–4890.
duction of high levels of IL-4 and IL-10 in response to the infec-
tions with this parasite. In contrast, C57BL/6 mice are resistant to
L. major infections and typically produce high levels of IFN-? but
no IL-4 and IL-10 (2–5). Although multiple avenues have been
explored to elucidate mechanisms of host susceptibility of BALB/c
mice, it still remains unknown whether events in Ag processing
pathway can change the course of disease in L. major infections in
BALB/c mice. It is also unclear why most CD4 T cells elicited in
the susceptible BALB/c mice recognize a single epitope 158–173
of one protein, Leishmania homolog of receptor for activated c-
kinase (LACK)3(6–9), when the parasite is known to express
?10,000 different proteins. It can be postulated that a stringent
selection of MHC class II-associated epitopes must occur during
pathogen processing within the Ag processing pathway (10–13).
Besides catalyzing the exchange of class II invariant chain pep-
tide (CLIP) with antigenic peptides within the groove of MHC
rotection from Leishmania major, an obligate intracellular
parasite, is mediated by CD4?T cells (1–3). BALB/c mice
are susceptible to L. major and are characterized by pro-
class II molecules, DM, itself a nonclassical MHC class II mole-
cule, functions as a peptide editor in selecting the repertoire of
peptides displayed by MHC class II molecules (10–15). Such se-
lection greatly influences the repertoire of responding CD4 T cells
(10, 11). The default pathway of Ag-specific T cell responses in
normal mice is characteristically focused on recognition of a lim-
ited number of immunodominant epitopes (11, 16). However, we
have shown before that the specificity of responding T cells in the
absence of DM-driven epitope editing in DM-deficient (DM?/?)
mice expands to additional epitopes not recognized by T cells elic-
ited in responses in the DM-sufficient mice (10, 11). We therefore
hypothesized that T cells recognizing newer epitopes, not dis-
played by infected APC in the wild-type mice, could be qualita-
tively different in their ability to elicit the type of effector (e.g.,
TH1 or TH2 or TH17 type) response (17, 18), and thus could lead
to a change in the outcome of infection in DM?/?BALB/c mice.
We tested this hypothesis by using the recently constructed
DM?/?BALB/c mouse (10, 19) (note that BALB/c embryonic
stem cells were used to construct DM?/?mice (19). Importantly,
the consequences of DM peptide editing function on immunity
against pathogens have remained unexplored primarily because the
DM-deficient mice were initially constructed in a C57BL/6 (H-2b)
haplotype (19, 20). Allelic properties of AbMHC (unlike Ad, Ed,
or AkMHC) encode a very tight CLIP-MHC association such that
in the absence of DM, all Abmolecules expressed by APC exclu-
sively display CLIP (19–21). This attribute of AbMHC precludes
the use of C57BL/6 mice for an exploration of the role of DM
peptide editing function in immunity (10, 22). However, Adand Ed
alleles can largely spontaneously dissociate from CLIP without the
assistance of DM (10, 19), and this allowed us to examine the
consequences of DM epitope editing in DM?/?BALB/c mice dur-
ing L. major infections.
Invariant chain (Ii) is another molecule, a chaperone, expressed
within the Ag processing pathway and is required for trafficking of
MHC class II from the endoplasmic reticulum to the endocytic
pathway within APC. Ii-deficient (Ii?/?) mice show a substantial
*Laboratory of Cellular and Molecular Immunology, T-Cell Tolerance and Memory
Section, National Institutes of Health, Bethesda, MD 20892; and †Medstar Research
Institute, Hyattsville 20873, MD and Department of Microbiology and Immunology,
Georgetown University Medical Center, Washington DC 20057
Received for publication November 25, 2008. Accepted for publication January 30,
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by Department of Defense (award W81XWH-04-0013; to N.K.N.)
and the Division of Intramural Research, National Institute of Allergy and Infectious
Diseases, National Institutes of Health.
2Address correspondence and reprint requests to Dr. N. K. Nanda, Laboratory of
Cellular and Molecular Immunology, Building 4, National Institutes of Health, Be-
thesda, MD 20892. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: LACK, Leishmania homolog of activated receptor
for c-kinase; BMDC, bone marrow-derived dendritic cell; CLIP, class II invariant
chain peptide; DC, dendritic cell; FP, foot pad; Ii, invariant chain; LN, lymph node;
7AAD, 7-aminoactinomycin D; SLA, soluble Leishmania Ag.
The Journal of Immunology
quantitative decrease (unrelated to epitope editing) in epitope dis-
play as a result of altered trafficking and decreased class II expres-
sion on APC. BALB/c Ii?/?mice were therefore included in our
studies to contrast the DM-mediated constraints in epitope display
vs quantitative change in presentation of epitopes in Ii?/?mice
(23, 24). Interestingly, Ii?/?mice also show allele-specific differ-
ential outcomes for Ag presentation in different haplotype mice.
Thus, although C57BL/6 Ii?/?(23) mice show severe Ag presen-
tation defects and marked decreases in the numbers of mature CD4
T cells in the thymus and the periphery, BALB/c Ii?/?mice show
only limited defects in Ag presentation function and have rela-
tively efficient maturation of CD4 T cells in the periphery (19, 23,
24). Thus, H-2dalleles can allow MHC class II trafficking and
peptide loading via an Ii-independent pathway. Furthermore, dou-
bly deficient DM?/?Ii?/?mice (19) were used to examine
whether DM deficiency can influence the outcome of disease when
not only is there a substantial quantitative decrease in epitope dis-
play but, additionally, peptide loading of class II takes place in the
absence of DM.
The results presented herein indicate that DM-deficient (DM?/?
and DM?/?Ii?/?) mice are resistant to infection with L. major. We
show that the resistance of BALB/c DM?/?and DM?/?Ii?/?
mice is attributable to elicitation of a distinctly different group of
T cells recognizing a newer array of epitopes that are not displayed
by APC in the infected wild-type BALB/c mice. The data also
show that presence of T cells specific for the newer epitopes in
DM-deficient mice leads to production of protective levels of
IFN-? accompanied by an absence of secretion of IL-4 or IL-10
through the entire course of disease. This report thus suggests that
protective responses can be elicited in BALB/c mice infected with
L. major when Ag processing takes place in the absence of DM.
Materials and Methods
BALB/c/J and C57BL/6 mice were purchased from The Jackson Labora-
tory. Homozygous mutant strains DM?/?, Ii?/?, and DM?/?Ii?/?(10, 19)
and the wild-type B10.D2 mice were bred in a specific pathogen-free fa-
cility. All mice were maintained under American Association for the Ac-
creditation of Laboratory Animal Care-approved conditions.
Parasites, infections, Ags, and peptides
L. major MHOM/IL/80/Friedlin (a gift from Dr. David Sacks, Laboratory
of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health) was cultured in M199 medium containing
20% FCS, and metacyclic promastigotes were purified from stationary
phase cultures using peanut agglutinin as described (25). Mice were
infected in the left hind footpad with 0.5 ? 106metacyclics in
Soluble Leishmania Ag (SLA) was prepared by homogenizing station-
ary phase parasites (5 days in culture), using seven freeze-thaw cycles, and
centrifuged at 10,000 rpm for 10 min at 4°C. Protein content was quantified
using the BCA protein assay reagent (Pierce), with freeze-thaw lysates of
100 ? 106parasites yielding 77 ?g of protein on average. Filter-sterilized
(0.22 ?m) lysate (SLA) was stored in 1-ml aliquots at ?80°C.
Peptide 158–173 was custom synthesized and purified by Macromolec-
ular Resources as described before (10).
Estimation of parasite load in infected tissues
Numbers of live parasites were quantified as described before (25). Briefly,
at various times after infection, infected footpads, a pool of draining pop-
liteal, inguinal, and periaortic lymph nodes (LN), liver, and spleen were
isolated and either weighed (in the case of foot pads and livers) or disso-
ciated for lymphocyte count (in the case of LN and spleen). Homogenized
tissue samples were plated in 96-well round-bottom plates at 25°C. For
limiting dilution analysis, four to eight replicates per sample were cultured
in 7–11 serial dilutions (5- to 10-fold each dilution) in M199 medium
containing 20% FCS. Visible parasite colonies were scored under the mi-
croscope at 3 wk. The parasite number was calculated from minimum ?2
analysis of the Poisson distribution relationship between dilution of ho-
mogenate and percentage of negative wells (i.e., wells that did not grow out
any parasites). We assumed that a dilution that had 37% negative wells
equated to at least one L. major CFU (26). Each symbol in Fig. 2 represents
data from one mouse.
Generation of L. major-specific hybridomas from DM?/?
Draining LN T cells from 4-wk-infected DM?/?BALB/c mice were cul-
tured at 5 ? 106cells per well in 24-well flat bottom plates with 100 ?g
of SLA. Five days later the T cell blasts were harvested and fused with
BW5147?-?-hybridoma partner cells using standard methodology as de-
scribed before (except that the current hybridomas were not subcloned)
(27). Ag specificity of hybridomas was assessed by IL-2 secretion response
following overnight culture of hybridomas with 24-h L. major infected or
uninfected DM?/?bone marrow-derived dendritic cells (BMDC).
LN or spleen cells were cultured at 5 ? 106cells per well in 24-well flat
bottom plates (Costar) in the presence or absence of 100 ?g of SLA. Cul-
ture supernatants obtained after 7 days (or after indicated times as in data
presented in supplemental Fig. 44for time kinetics) were analyzed for IL-4,
?-IFN, IL-10, and IL-17 by sandwich ELISA using commercial kits (R&D
Systems) and as described before (11).
Infection of BMDC with L. major
Bone marrow (BM) cells from wild-type and mutant BALB/c mice were
harvested from long bones of thighs. Cells were frozen at 5 ? 106cells/ml
and stored in liquid nitrogen until needed. BMDC were grown using a
variation of a previously published protocol (28). BM cells were thawed
and plated at 1 million cells per ml per well in 24-well plates in advanced
DMEM-F12 (Invitrogen) supplemented with 2 mM glutamine, 5% FCS, 50
?g/ml gentamicin, 100 U/ml penicillin, 100 ?g/ml streptomycin and 12.5
?M 2-ME, and 6 ng/ml GM-CSF and 3 ng/ml IL-4 (both from PeproTech).
On day 6, live stationary phase L. major promastigotes were added to
BMDC cultures at 35 ? 106parasites per well. One day later, infected
BMDC cultures were either used for cytospin preparations or for epitope
display (see below). BMDC, cultured as above, and stained with anti-
CD11b (clone M1/70), anti-CD11c (clone N418), and anti-MHC class II
(clone M5/114) Abs and 7AAD (7-aminoactinomycin D) were 90–95%
CD11c?MHC class II-high cells. Immunofluorescence data were acquired
on a BD FACSort cytometer and analyzed using Cell Quest v3.3 (BD
Immunocytometry Systems). All Abs used in this study were from BD
Biosciences, unless stated otherwise.
Cytospins of uninfected and infected BMDC (infected as above) were
stained with Giemsa (Histoserv) and digitally photographed using a Zeiss
Axiophot microscope at ?100 magnification to analyze their degree of L.
major infection. Images were processed with IPLab 3.5 and Photoshop 3.0
Epitope display by L. major-infected BMDC from BALB/c
wild-type and mutant mice
Replicate cultures of 2-fold serial dilutions (375–192,000) or indicated
numbers of WT, Ii?/?, DM?/?, and DM?/?Ii?/?BMDC of washed in-
fected (as above) and uninfected BMDC were either not pulsed or pulsed
with 1 ?M LACK peptide 158–173 and cultured with 100,000 cells per
well of LMR 16.2 hybridoma cells (specific for LACK peptide 158–173;
a gift from Dr. N. Glaichenhaus, Institut National de la Sante ´ et de la
Recherche Me ´dicale, Nice, France). After overnight culture, supernatants
were analyzed for IL-2 by ELISA performed according to the manufac-
turer’s instructions (R&D Systems).
In vivo anti-IFN-? treatment
Anti-IFN-? (clone XMG1.2) and isotype control Ab (clone HRPN) were
purchased from Bio X Cell. Mice were injected with 500 ?g of Ab in 200
?l i.p. weekly for 5 wk.
BALB/c DM?/?and DM?/?Ii?/?mice show protection against
L. major infections
To assess whether Ag processing in the absence of DM could alter
the course of L. major infection in BALB/c mice, we infected
4The online version of this article contains supplemental material.
4883 The Journal of Immunology
groups of wild-type, DM?/?, Ii?/?, and DM?/?Ii?/?BALB/c
mice with 0.5 ? 106metacyclic parasites in the foot pad (FP) and
measured FP size to determine disease progression. Fig. 1 shows
the size of L. major-infected FP in mice during 15 wk after infec-
tion for each group from one representative of five experiments. As
expected, the FP size in susceptible wild-type (DM?/?) BALB/c
mice (Fig. 1A) progressively increases with time until it was nec-
essary to sacrifice them. Strikingly, however, the infected DM?/?
mice showed only a minimal increase in FP size (Fig. 1B). FP
swelling in the infected feet of Ii?/?mice nearly paralleled those
of the wild-type mice in that they exhibited progressive swelling at
most time points (Fig. 1C). Remarkably, however, the DM?/?
Ii?/?mice showed a FP swelling pattern that was similar to that of
the resistant DM-deficient mice during the entire 15 wk postinfec-
tion period (Fig. 1D). Thus, while the infected wild-type (5) and
Ii?/?(29) BALB/c mice were susceptible as expected, the DM?/?
and DM?/?Ii?/?BALB/c mutant mice resisted the development
of any noteworthy or sustainable FP lesions.
DM?/?and DM?/?Ii?/?mice control local parasite load and
are protected against systemic parasite spread
We next explored whether L. major resistance of DM?/?and
DM?/?Ii?/?mice was due to clearance of parasites from the site
of infection (FP) and/or from spleen and liver, the typical sites of
systemic spread in the wild-type mice (30), by evaluating the par-
asite loads in these tissues of infected mice. Fig. 2 (bottom panels)
shows that the parasite loads in the FP of the susceptible wild-type
and Ii?/?mice increased exponentially during the course of in-
fection. In contrast, the parasite numbers in FP of DM?/?and
DM?/?Ii?/?mice were significantly lower and continued to con-
sistently decline until the average parasite number was only 800
and 100, respectively, by day 90. These data demonstrate a control
are protected from systemic parasite spread and clear
parasite load in FP. Age-matched, BALB/c/J wild-type,
DM?/?, Ii?/?, and DM?/?Ii?/?were infected in one
hind FP as described in Fig. 1 and in Materials and
Methods. Three to five mice from each group were sac-
rificed at the time points indicated postinfection. The
data shown are from individual mice from two to three
different experiments. FP, spleens, and livers were ho-
mogenized and cultured in limiting dilution and the par-
asite numbers were calculated as described before (25,
26) and in the supplemental materials. Horizontal bars
represent the geometric means of data at a given time
point. All wild-type and the Ii?/?mice had to be sac-
rificed at day 52 due to their large FP sizes except one
rare wild-type mouse that we were able to analyze on
day 78 postinfection. Note that the DM?/?and
DM?/?Ii?/?BALB/c mice have cleared parasites in
spleens and livers and have controlled parasite num-
bers to between 100 and 800 in the FP by day 90
postinfections. Wild-type (F), Ii?/?(Œ), DM?/?(f)
DM?/?and DM?/?Ii?/?BALB/c mice
are resistant to FP infection with L. major. BALB/c/J,
DM?/?, Ii?/?, and DM?/?Ii?/?were infected in one
hind FP with 0.5 ? 106metacyclic promastigote L. ma-
jor parasites. Twelve to 18 age-matched female (two
experiments) or male and female (three experiments)
mice were used for each group. The thickness and di-
ameter of the FP was measured each week postinfection.
Closed symbols represent the sizes of the infected FP
and the open symbols represent sizes of the uninfected
foot pads. Note that the lesion size in the wild-type and
Ii?/?mice continued to grow and these mice had to be
euthanized (E) by day 84. In contrast, the DM?/?and
DM?/?Ii?/?mice developed minimal lesions and the
FP size of these mice was measured until day 105
postinfection. Note that in our experience the time point
at which the wild-type mice reached maximal FP lesions
varied between week 6 and 12 in different experiments.
Mice infected with 2.0 ? 106parasites showed the same
DM?/?and DM?/?Ii?/?BALB/c mice
4884DM?/?BALB/c MICE ARE PROTECTED AGAINST L. MAJOR INFECTIONS
of infection in FP of both the DM?/?and DM?/?Ii?/?mice
Systemic spread of parasites to visceral sites such as spleen and
liver is one of the hallmarks of FP L. major infection in BALB/c
mice (30). Accordingly, the spleens and livers of infected wild-
type and Ii?/?mice showed sustained parasite presence and an
exponential increase in parasite numbers until the last day before
being sacrificed (Fig. 2, top and middle panels). Contrastingly,
however, not only did the spleens and livers of infected DM?/?
and DM?/?Ii?/?mice generally show remarkably lower numbers
of parasites at all time points examined (an average of 300 and 9
in spleens and of fewer than 10 parasites in livers of DM?/?and
DM?/?Ii?/?mice, respectively, on day 54 postinfection), they
had completely cleared the infection by day 90 (Fig. 2, top and
middle panels). These data in DM-deficient mice are strikingly
similar to those obtained for the systemic parasite loads in the
healer strain C57BL/6 (see supplemental Fig. 1). Our results
strongly indicate that L. major is not able to establish and/or sus-
tain systemic infection in DM?/?and DM?/?Ii?/?mice. Addi-
tionally, to ensure that resistance of DM?/?and DM?/?Ii?/?
mice was not a result of an unknown intrinsic defect caused by
deficiency of DM preventing L. major from infecting the mutant
APC, we infected BMDC from wild-type and mutant (DM?/?,
Ii?/?, and DM?/?Ii?/?) BALB/c mice in vitro with L. major.
Giemsa staining of infected dendritic cells (DC) (indicates that L.
major small intracellular black dots in Fig. 3) is able to infect
wild-type and mutant DC in a comparable manner. Furthermore,
the data shown in Fig. 2 indicate that the parasite load at the site
of infection (FP) is not very different in the wild-type and the
mutant strains of mice at early time points postinfection, providing
additional evidence that infectivity of L. major for the different
mutants is comparable to that of the wild-type mice.
L. major-infected APC from resistant BALB/c DM-deficient mice
display a different set of epitopes as compared with infected
wild-type and Ii?/?APC
Two different strategies were used to explore the effects of DM-
mediated epitope editing on the display of Leishmania epitopes by
L. major-infected APC in wild-type vs DM-deficient strains. It is
well established that most T cells elicited in L. major infected
wild-type BALB/c mice only recognize a single immunodominant
epitope, LACK 158–173/Ad. Thus, in the first approach, we used
the wild-type BALB/c-derived (LACK 158–173-specific) T cell
hybridoma to probe the expression of this epitope on infected DM-
deficient APC. Contrastingly, in the second approach, we devel-
oped a panel of L. major-specific T cell hybridomas from infected
DM?/?mice and used them as probes to explore whether identical
epitopes were expressed by the infected wild-type APC.
L. major infected DM?/?APC fail to display the immunodominant
epitope LACK 158–173. Wild-type BALB/c-derived LACK 158–
173-speciifc T cell hybridoma LMR 16.2 was used as a probe to
examine the display of the immunodominant epitope LACK 158–
173 by infected DC from the wild-type, DM?/?, Ii?/?, and DM?/?
Ii?/?mice. As expected, L. major-infected DC from wild-type
BALB/c mice efficiently displayed the immunodominant LACK
epitope, and rather low numbers (n ? 375) of infected APC were
able to stimulate IL-2 production from the T cell hybridoma (Fig.
4, top left panel). In sharp contrast, infected DM?/?DC did not
stimulate the LACK 158–173-specific hybridoma even at the high-
est numbers (n ? 192,000) of infected DC per well (Fig. 4, right
upper panel). As a consequence of abnormal trafficking of MHC
class II in the absence of Ii, it can be expected that infected Ii?/?
and DM?/?Ii?/?APC (19) will display quantitatively low levels
of parasite-derived epitopes (19, 24). Accordingly, APC from
Ii?/?DC were able to stimulate LACK 158–173-specific hybrid-
oma but only when 48,000–192,000 infected DC were used in the
culture well (Fig. 4, bottom left panel). These data reflect a definite
though quantitatively scarce display of epitope 158–173 by in-
fected Ii?/?APC (19). Interestingly, however, DM?/?Ii?/?APC
failed to stimulate LACK 158–173-specific T hybridoma even at
the highest numbers of infected DC (Fig. 4, right lower panel).
This result suggests that the expression of low amount of LACK
158–173 in Ii?/?APC was dependent upon presence of DM.
Note that the infected or uninfected DC from all mice including
DM?/?Ii?/?mice were able to show activation of LMR 16.2
when exogenous peptide 158–173 was added to the culture wells
(Fig. 4, insets), indicating that levels of expression of MHC class
II and costimulatory molecules in all DC were sufficient to stim-
ulate T cell hybridomas. Data in Fig. 4 thus indicate that display of
MHC class II-bound LACK 158–173 is completely abrogated in
L. major-infected DM?/?APC present newer epitopes not dis-
played by infected wild-type APC. As mentioned above, a panel of
16 L. major-specific T cell hybridomas was derived from infected
BALB/c DM?/?mice 4 wk postinfection (see Materials and
Methods). All 16 DM?/?-derived hybridomas showed IL-2 secre-
tion only when L. major-infected DC were used as APC but no
DM?/?, Ii?/?, and DM?/?Ii?/?DC
are infected by L. major in a compa-
rable manner. BMDC, prepared as de-
scribed before (28) and in Materials
and Methods, were infected with
35 ? 106L. major promastigotes (at a
DC/parasite ratio of 1:35) for 24 h.
Cytospin slides prepared from in-
fected and uninfected BMDC were
stained with Giemsa (as described in
Materials and Methods) and photo-
graphed (shown at ?100 magnifica-
tion). Note that the small black
(darkly stained) intracellular dots rep-
resent L. major nuclei (and the large
black organelle is the DC nucleus).
4885The Journal of Immunology
IL-2 production when uninfected DC were used as APC (Fig. 5A).
The peptide specificity of these 16 hybridomas is unknown (pre-
liminary analyses (not shown) indicate that these DM?/?-derived
hybridomas may not be reactive to LACK-derived peptides; how-
ever, our data do not entirely rule out this possibility).
Nevertheless, we used these hybridomas to explore whether
identical epitopes were being displayed by L. major-infected
DM?/?and wild-type APC. As shown in Fig. 5A, while 15 of 16
hybridomas were stimulated by epitopes displayed by infected
DM?/?APC (right panel), only 7 of 16 hybridomas were stimu-
lated by L. major-infected wild-type APC (left panel). Thus, eight
different L. major-specific hybridomas were unable to recognize
any epitope presented on infected wild-type DC. Dose response of
infected wild-type DC shown in Fig. 5B confirms that infected
wild-type APC completely lack presentation of these epitopes for
hybridomas 2E5 and 1F7, as there is no secretion of IL-2 even
when they are stimulated by 192,000 infected DC. These data af-
firm that relative to the wild-type-infected APC, 1) at least one
(and very likely more than one) newer pathogen-derived epitope is
displayed by L. major-infected DM?/?APC, and 2) the display of
these newer pathogen epitopes is quantitatively sufficient for acti-
vation of a distinct repertoire of T cells in DM?/?mice in response
to L. major infections. To precisely state the diversity of newer
epitopes displayed by infected DM?/?APC but not by the simi-
larly infected wild-type APC, we await identification of peptides
recognized by these eight distinct hybridomas.
Furthermore, 7 of 15 hybridomas recognize epitopes displayed
on L. major-infected wild-type and DM?/?DC (Fig. 5A). A dose
response of one of these T hybridomas, 2D8, is shown in Fig. 5B.
Since infected DM-deficient APC lack a display of LACK 158–
173 (Fig. 4), LACK 158–173 epitope can be precluded as an
epitope recognized by this set of seven hybridomas. Our prelimi-
nary data (not shown) affirm this prediction. The data, however,
dominant epitope LACK 158–173. Bone marrow DC, prepared from 1 ?
106BM cells as described before (28) and in Materials and Methods, were
infected with 35 ? 106L. major promastigotes for 24 h. Infected and
uninfected DC were washed and used at various concentrations with the T
cell hybridoma (LMR 16.2) specific for LACK 158–173. The data are
depicted as IL-2 release, a measure of activation of the T cell hybridoma,
and are representative of three to five experiments. Peptide 158–173 (1
?M) was exogenously added in the data shown in the insets. Open symbols
represent uninfected and closed symbols represent infected BMDC: Wild-
type (F, E), DM?/?(f, ?), Ii?/?(Œ, ‚), and DM?/?Ii?/?(?, ƒ).
L. major-infected DM?/?APC fail to display the immuno-
displayed by infected wild-type DC. A, Sixteen L. major-specific hybridomas
derived from infected DM?/?mice were examined for activation using L.
major-infected or uninfected wild-type or DM?/?BMDC (104DC per well)
as described in Fig. 4. The data show that while 15 of the 16 DM?/?-derived
hybridomas responded to infected DM?/?APC, only 7 of 16 recognized in-
fected wild-type APC, thus indicating that L. major-infected wild-type DC
failed to express epitopes recognized by 8 DM?/?T cell hybridoms. The
horizontal dotted line represents the cutoff point for the T hybridomas not
recognizing L. major infected APC (with an exception of 1C3, which shows a
(E), infected (F); BALB/c DM?/?uninfected (?), infected (f). B, Three
DM?/?-derived T hybridomas were examined for stimulation with varying
doses of uninfected or infected BMDC as described in A. The data show that
2E5 and 1F7 hybridomas (representing the eight T cell hybridomas that fail to
be stimulated by infected wild-type APC) are not stimulated despite the pres-
ence of 192,000 infected wild-type DC per well. The hybridoma 2D8 (repre-
senting seven T hybridomas that are stimulated by both the infected wild-type
and DM?/?DC) shows an enhanced response to wild-type DC as compared
with infected DM?/?DC. Symbols used are as in A.
L. major infected DM?/?DC present newer epitopes not
4886DM?/?BALB/c MICE ARE PROTECTED AGAINST L. MAJOR INFECTIONS
suggest that 2D8 hybridoma responds to an epitope that can be
presented in the absence or in the presence of DM.
DM?/?and DM?/?Ii?/?mice produce IFN-? and IL-17 but
To explore whether the effector cytokine response of the resistant
DM?/?and DM?/?Ii?/?mice matched that of the healing strain
C57BL/6, we examined the cytokines produced by LN cells from
infected DM?/?and DM?/?Ii?/?(as well as by the wild -type and
Ii?/?mice) at various time points postinfection (Fig. 6A). As ex-
pected, the wild-type and Ii?/?mice produced high levels of IL-4
and IL-10 (accompanied by modest amounts of IFN-?) (2–5, 31).
Strikingly, however, BALB/c DM?/?and DM?/?Ii?/?mice
showed no production of IL-4 or IL-10 in response to L. major
infections at any time point, yet they produced IFN-? throughout
the course of disease (see Fig. 6A, right panels). Lack of IL-4 and
IL-10 production in protected DM-deficient mice matched the ab-
sence of IL-4 and IL-10 production in the healer C57BL/6 mice
(32–34) (supplemental Fig. 2).
The ratio of IFN-?/IL-4 depicted in Fig. 6B suggests that the
strikingly higher ratio of IFN-? to IL-4 in DM?/?and DM?/?
Ii?/?mice parallels resistance, and the lower ratio corresponds to
susceptibility for both the wild-type and Ii?/?mice. These results
support the previously held conviction that in the absence of the
inhibitory cytokines IL-4 and IL-10, even low amounts of IFN-?
are sufficient to activate macrophages to produce inducible NO
synthase and other effector molecules necessary for parasite clear-
ance (33). The absence of IL-4/IL-10 in DM?/?and DM?/?Ii?/?
strains should therefore allow relatively potent activation of DC
and parasite clearance (33, 35).
We also measured levels of IL-17 (another proinflammatory
cytokine) in infected mice and show that the resistant DM?/?,
DM?/?Ii?/?, and C57BL/6 mice produced IL-17, especially at the
late stage of disease (Fig. 6A and supplemental Fig. 2). In contrast,
IL-17 was not produced by wild-type mice at any time point
postinfection. Nevertheless, a markedly high ratio of IL-17 to IL-4
symbolizes protection in DM?/?and DM?/?Ii?/?(Fig. 6B, right
panel) and C57BL/6 mice (not shown), and a low ratio correlates
(shown) and spleen (not shown) cultured in vitro with SLA for 7 days were analyzed for a panel of cytokines using ELISA technique as described in Ref.
11. Time kinetics of cytokines expressed in supernatants from LN cells cultured for 24, 48, 72, 96, and 168 h are shown in supplemental Fig. 4. The data
in supplemental Fig. 4 validate our choice of a 7-day period of in vitro culture. The data from individual mice from three to nine mice per strain per time
point are shown. The horizontal lines represent median values of each group. The pattern of cytokine secretion from spleen cells (not shown) was similar
to that from lymph node cells. Wild-type (F), DM?/?(f), Ii?/?(Œ), and DM?/?Ii?/?(?). B, DM?/?and DM?/?Ii?/?mice have significantly higher
ratios of IFN-? vs IL-4 and IL-17 vs IL-4. Data from A are shown as ratio of average value of IFN-? vs IL-4 (left panel) and of IL-17 vs IL-4 (right panel)
at a given time point postinfection to indicate that the resistance phenotype of BALB/c DM?/?and DM?/?Ii?/?mice is correlated with markedly higher
ratios of inflammatory cytokines IFN-? (left panel) and IL-17 (right panel). Color of bars are as in A. C, DM?/?mice do not make IL-4 and IL-10 even
7 days postinfection. The supernatants derived from LN cells derived from mice infected 7 days prior were cultured in vitro with SLA and were analyzed
for a panel of cytokines as described in A. Data from seven to nine mice are shown as in A. GraphPad Prism 5 for MAC OS X was used for unpaired t
test analysis in C. ?, p ? 0.05; ??, p ? 0.005. Symbol colors are as in A. Open symbols indicate no Ag (insets); closed symbols, with SLA.
A, DM?/?and DM?/?Ii?/?mice do not produce IL-4 and IL-10 in response to L. major infections. The supernatants derived from LN cells
4887The Journal of Immunology
with susceptibility of DM?/?mice. Note that production of IL-17
by infected DM-deficient mice was neither just exclusive to L.
major infections nor was it produced in an Ag-nonspecific manner.
LN cells from hen egg lysozyme-immunized DM?/?and the wild-
type mice showed equivalent production of IL-17 in response to
the immunodominant HEL 106–116 but none when cultured with-
out Ag (data not shown).
To determine exactly how early after infection with L. major
divergence of cytokine in DM-deficient mice vs the wild-type mice
could be observed, we examined cytokine secretion in infected
mice on day 7 postinfection (Fig. 6C). It is clear that the hallmark
cytokine response of no IL-4/IL-10 but secretion of IFN-? and
IL-17 in resistant DM?/?, DM?/?Ii?/?(Fig. 6C), and B6 mice
(supplemental Fig. 2, bottom panels) was already in existence as
early as day 7 postinfection. The pattern of high IL-4/IL-10 re-
sponse observed in the susceptible strains of mice was also already
existent at day 7 (Fig. 6C). Furthermore, our preliminary data in-
dicate that this characteristic cytokine response of DM- deficient
mice vs the susceptible DM-sufficient mice is detectable on day 2
postinfection (not shown). These results suggest that pathogen pro-
cessing in DM-deficient mice triggers events (such as an activation
of a newer group of T cells in place of LACK 158–173-reactive T
cells) that can steer the host toward protective immune responses
as early as day 2 postinfection. We propose that this early differ-
ential cytokine response between the susceptible wild-type and the
resistant DM-deficient mice underlies the presence and absence,
respectively, of FP swelling in these strains shown in Fig. 1.
Anti-IFN-? Ab treatment abrogates protection of
BALB/c DM?/?mice against L. major infections
To explore whether resistance of DM-deficient mice was indeed
due to IFN-? production and a high IFN-?/IL-4 ratio, we treated
DM?/?and wild-type BALB/c mice weekly with 500 ?g of anti-
IFN-? or an isotype control Ab for 5 wk. Mice were infected with
L. major a day after the first injection with the Abs. B10.D2 and B6
mice, treated with the same regimen of Abs and infection, were
included as controls. As shown in Fig. 7, all DM?/?mice treated
with anti-IFN-? Ab showed remarkable susceptibility to infections
(measured by the FP swelling) and had to be euthanized by day 35
postinfection (top panels). Contrastingly, as expected (Fig. 1),
DM?/?mice that received isotype control Ab were protected.
Wild-type mice treated with IFN-? Ab developed a more severe
disease (larger FP swelling) relative to those treated with the iso-
type control (Fig. 7, see insets in bottom panels). All of the nor-
mally resistant B10.D2 (Fig. 7, bottom panels) and B6 mice (sup-
plemental Fig. 3) treated with anti-IFN-? also became susceptible
to infections, whereas those treated with isotype control Ab re-
mained resistant to infections. These results demonstrate that the
resistance to L. major in DM-deficient mice is dependent on IFN-?
produced in response to infection.
We identify DM as a host susceptibility gene for L. major infec-
tions in BALB/c mice since the presence of DM leads to suscep-
tibility and its absence leads to host protection against this parasite.
Paradoxically, the opposite is true in the case in C57BL/6 mice. As
mentioned before, unlike Adand Ed, AbMHC encodes a very tight
CLIP-MHC association, and in the absence of DM, all Abmole-
cules expressed by APC exclusively display CLIP (19, 20, 21).
This attribute of Abmakes DM a host resistance gene in B6 mice
since DM-deficient B6 mice, unable to make any pathogen-specific
CD4 T cell response, become susceptible to L. major infections
(22). These data highlight how molecular events in pathogen pro-
cessing can profoundly alter the course of susceptibility to parasite
BALB/c response to L. major infection is characterized by very
high levels of IL-4 and IL-10 production (5), immunodominance
of the LACK 158–173 epitope (9), and systemic spread of the
parasites from the skin (the site of infection) to visceral sites such
as spleen and liver (30). We show herein that ablation of the DM
molecule in BALB/c mice also ablates presentation of the immu-
nodominant LACK 158–173 epitope and in its place allows dis-
play of newer epitopes (one or more) not presented by DM-suffi-
cient, wild-type infected APC (Fig. 5). Although the identity of the
newer peptides displayed by DM deficient APC is unknown, our
preliminary data (not shown) indicate that they may not be derived
from LACK. The presentation of the newer epitopes, not displayed
by the wild-type APC, is quantitatively sufficient to have activated
CD4 T cells leading to isolation of 16 L. major-specific T cell
hybridomas from infected DM?/?mice. We show that these
events, including an ablation of presentation of LACK158–173
and activation of a newer set of T cells specific for epitopes ex-
cluded from presentation in wild-type mice, have profound immu-
nological consequences in that the DM?/?mice are now resistant
to L. major infection and are capable of controlling systemic
Groups of five BALB/c wild type and DM?/?mice each and three B10.D2
mice each were injected i.p with 500 ?g of anti-IFN-? Ab or with isotype
control Ab weekly for 5 wk starting 1 day before FP infection with 0.5 ?
106L.major as described in Fig. 1. FP swelling was measured over the
course of 55 days and is depicted as shown in Fig. 1. Closed symbols
represent the sizes of the infected FP and open symbols represent sizes of
the uninfected FP. All of the IFN-?-treated DM?/?, wild-type BALB/c
mice and two of three IFN-?-treated B10.D2 mice and all of the isotype
Ab-treated wild-type BALB/c mice had to be sacrificed between day 35
and 44 due to animal protocol stipulations. Due to strikingly similar
results in all individual mice in each group, this particular experiment
was done once.
IFN-? is essential for controlling disease in DM?/?mice.
4888DM?/?BALB/c MICE ARE PROTECTED AGAINST L. MAJOR INFECTIONS
spread of parasites, and the immune response consists of protective
levels of IFN-? and IL-17 and an absence of IL-4 or IL-10.
How does Ag processing in the absence of DM lead to a diver-
gence in effector cytokine response in BALB/c mice in response to
L. major infections? Effector cytokines are secreted by L. major-
specific host CD4 T cells. As mentioned above, most CD4 T cells
isolated from wild-type BALB/c mice recognize LACK 158–173.
In contrast, CD4 T cell response in L. major-infected DM?/?mice
is represented by 1) an absence of LACK 158–173-specific CD4 T
cells, and 2) the specificity of eight L. major-specific DM?/?-
derived CD4 T cell hybridomas for epitopes exclusively displayed
by DM?/?APC and precluded from epitopes presented by in-
fected wild-type APC (Fig. 5). We propose that a switch to a
protective response (secretion of both IFN-? and IL-17) in DM?/?
mice is a consequence of the absent LACK 158–173-specific T
cell response coupled with elicitation of T cells specific for newer
epitopes that were precluded from presentation in DM-sufficient
APC. It is expected that T cells specific for different epitopes
would be nonidentical in their ability to elicit different effector type
response (e.g., TH1 or TH2 or TH17 type) based on their avidity
interactions and strength of signaling induced in the target T cell
(17, 18). Thus, it can be speculated that avidity interactions en-
coded within the newer epitopes presented in absence of DM lead
to IFN-? response and lack of IL-4/IL-10 response, while the op-
posite may be the case for LACK 158–173 response of a high
IL-4/IL-10 production. However, note that in the case of the im-
munodominant LACK 158–173, elicitation of high IL-4/IL-10 re-
sponse has also been postulated to be the result of a preexistent
memory response thought to be due to a cross-reactive epitope
expressed by a previously encountered common pathogen (36).
Such a memory response might be absent from DM?/?mice as a
consequence of the absent presentation of the putative LACK
158–173 cross-reactive epitope.
How does one view the epitopes displayed by both the wild-type
and DM-deficient APC in the context of the well-established im-
munodominant response LACK 158–173 in the wild-type mice in
response to L. major infections? We anticipate that the immuno-
dominant presentation of LACK 158–173 might mask the presen-
tation of other minor epitopes or/and that T cells specific for
epitopes other than LACK 158–173 might have been deleted as a
result of a differential negative selection in the wild-type BALB/c
mice. Although both possibilities could occur, evidence for one of
these possibilities was reported in our earlier work in which it was
shown that LACK 33–48-specific T cells could not be elicited in
the wild-type BALB/c mice despite immunization with the peptide
33–48, which requires no Ag processing (10). In contrast, the DM-
deficient mice immunized with the same peptide (or LACK pro-
tein) were able to show activation of these T cells. From the per-
spective of a differential T cell repertoire potentially underlying a
differential outcome of disease, it is pertinent to note the differ-
ences in TCR repertoires of the susceptible wild-type BALB/c
(self-superantigens Mls-2a, Mls-3a) and the normally resistant
B10.D2 (self-superantigens Mls-2b, Mls-3b) mice due to expres-
sion of different self-superantigens. We found that although TCR
V?5, V?11, and V?12 are completely deleted in BALB/c mice,
the same TCR gene segments are only partly deleted in B10.D2
mice (data not shown). Furthermore, unlike their BALB/c coun-
terparts, the B10.D2 mice have an intact repertoire of TCR V?3
gene segments (37). It can thus be speculated that the different
TCR repertoire available in B10.D2 mice could in part play a role
in the resistance of these mice to L. major infections as a conse-
quence of activation of T cells that recognize other epitopes (be-
sides LACK 158–173) displayed by (DM-sufficient) B10.D2 APC
and that produce protective cytokines.
Due to the deleterious nature of the immunodominant response
in the wild-type BALB/c mice thought to underlie the strong IL-
4/IL-10 and varying levels of IFN-? (2, 6, 36, 38) seen in response
to L. major infections, a number of studies have attempted to oblit-
erate the typical LACK 158–173-specific T cell response of in-
fected wild-type BALB/c mice by either inducing tolerance to this
epitope or by engineering the parasite to deliver an altered version
of LACK 158–173 peptide ligand to prevent activation of LACK
158–173-specific T cells (9, 36, 38). The results of these studies
have been ambiguous in terms of achieving resistance to L. major.
Our results suggest that the ambiguity of these studies might be
due to a lack of elicitation of the protective T cells in the wild-type
mice since DM-sufficient APC predominantly express the im-
munodominant Leishmania epitopes, and presentation of other
epitopes is either reduced or precluded. Similarly, an obliteration
of the nonprotective LACK 158–173 response in some of these
cases could have been incomplete while in others it might have
allowed some elicitation of a protective response. Note that
BALB/c Ii-deficient hosts display scarce levels of LACK 158–173
on APC and yet produce detrimental levels of IL-4/IL-10 and are
susceptible (Figs. 1–6). The present study shows that a require-
ment for protection of BALB/c hosts is dependent on T cells spe-
cific for the nonimmunodominant epitopes to produce the modest
amount of IFN-? required for protection. This is further affirmed
by our preliminary data (not shown) showing production of sig-
nature cytokines IFN-? and IL-17 in short-term (4–6 wk) cell lines
derived from infected DM?/?mice in response to L. major-in-
fected DC. The role of IL-17 in protection is less clear. However,
a high ratio of IFN-? to IL-4 and IL-17 to IL-4 production sym-
bolizes protective responses in DM-deficient mice (Fig. 6B). In-
terestingly, a robust innate immune response activation is seen in
L. major-infected DM?/?mice as indicated by significantly higher
production of IL-12p40 (supplemental Fig. 5). How ablation of
DM leads to greater activation of innate responses is unclear.
A recent study in MHC class I pathway showed ERAAP (the
endoplasmic reticulum aminopeptidase associated with Ag pro-
cessing) to be associated with resistance to Toxoplasma gondii
(39). Unlike our study, protection against the parasite was depen-
dent on expression of an immunodominant response to the para-
site. Taken together, these studies highlight that events in Ag pro-
cessing pathway are consequential for host protection against
pathogens. We propose that strategies to down-regulate and/or an-
tagonize DM function will be successful in protecting wild-type
BALB/c hosts against L. major. Development of agents that act as
antagonists for HLA-DM function could also be potentially used to
treat susceptibility to human infectious disease when the MHC
class II alleles dissociate from CLIP with relative ease.
We thank Drs. Ronald Schwartz and Polly Matzinger (National Institutes
of Health) for the generous and crucial support that made this work pos-
sible. We are grateful to Dr. Elizabeth K. Bikoff for supplying us with
breeder pairs for all of the BALB/c mutant mice, and May Awkal, Andrew
Heitman, Abhi Bhirud, Love Wade, and Dave Mallon for their excellent
technical assistance. We extend our special thanks to Dr. David Usharauli
for his suggestions. We are thankful to Drs. Ronald Schwartz, Polly Matz-
inger, David Usharauli, David Ucker, David Sacks, Elizabeth K. Bikoff,
and Nevil Singh for a critical reading of this manuscript.
The authors have no financial conflicts of interest.
4889 The Journal of Immunology
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