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Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France;
Institut National de la Santé et de la Recherche Médicale Unité 343, Nice, France;
Service de Bactériologie, Hôpital de l’Archet, Nice, France; and
Centre d’Immunologie Pierre Fabre, Saint Julien en Genevois, France.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2 and mediate the control of pathogen
replication and resistance to reinfection (1).
Although parasite-specific CD4+ T cells can be
detected in infected mice, little is known about their specificity and
their kinetics of activation and expansion. Early studies have
demonstrated that L. major induces the expansion of
CD4+ T cells which express the TCR V
8 and
V
4 variable regions (2). Accordingly, a significant
fraction of parasite-specific T cell clones which were isolated from
infected BALB/c mice used V8 and V4, suggesting that a single Ag
was the focus of the early immune response directed to the parasite.
This latter hypothesis was confirmed by the cloning of the
LACK5 Ag and the
demonstration that LACK-specific CD4+ T cells
used V8 and V4 (3).
Indirect evidence suggests that LACK-specific
CD4+ T cells are rapidly activated to secrete
IL-4 in infected animals. Thus, both L. major promastigotes
and recombinant LACK Ag induced the early and transient accumulation of
IL-4 transcripts within a subset of CD4+ T cells
that expressed V8 and V4 (4). This burst of IL-4
mRNA peaked in the draining LN of BALB/c mice 16 h after infection
with L. major or s.c. injection of LACK. Moreover, a burst
of IL-4 mRNA was also detected in the spleen of BALB/c mice 90 min
after i.v. injection of LACK (4).
Previous studies have demonstrated that naive and memory
CD4+ T cells exhibit distinct patterns and
kinetics of cytokine secretion. Thus, naive CD4+
T cells secrete mainly IL-2 on initial stimulation, whereas memory
CD4+ T cells synthesize a broader range of
cytokines, including IFN- and IL-4 (5, 6). Although
both naive and memory CD4+ T cells secrete IL-4
after in vitro stimulation, naive CD4+ T cells
require at least 36 h to express IL-4 mRNA, whereas memory T cells
synthesize IL-4 transcripts as early as 12 h after stimulation
(5, 6). Because LACK-specific T cells accumulate IL-4 mRNA
very rapidly in mice infected with L. major, we sought to
characterize their phenotype in BALB/c mice before infection. Here, we
demonstrate that the lymphoid organs of naive BALB/c mice contain
microbial Ag-specific T cells that cross-react to LACK and that express
a memory/effector phenotype. This phenomenon may account for the
ability of the LACK-specific T cells to rapidly secrete IL-4 shortly
after infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c and BALB/c-ByJ nude mice were purchased
from IFFA Credo (L’Arbresle, France). All animals were used between 6
and 8 wk of age. Mice were bred in our core animal facility and kept
under specific pathogen-free (SPF) conditions. Germfree (GF) BALB/c
mice were originally obtained from Dr. E. Balish and were maintained in
isolators with sterile food and water. When indicated, mice were
treated p.o. daily with 200 µl of a mixture of antibiotics containing
kanamycin (4 mg/ml), gentamicin (0.35 mg/ml), colistin (8500 U/ml),
metronidazole (2.15 mg/ml), and vancomycin (0.45 mg/ml). Treatment was
started at 2 wk of age. Surveillance for bacterial contamination was
performed by periodic bacteriologic examinations of feces
(7). In experiments in which mice were infected with
L. major, the treatment with antibiotics was stopped 1 day
before infection. DO.11.10 (8), TCR-LACK C°
(9), and IE-LACK transgenic mice have been described
(10).
Parasites and infection
L. major (World Health Organization strain WHOM/IR/-/173) promastigotes were maintained in M199 medium containing 20% FCS as described (3). Unless otherwise stated, mice were infected in the footpads with the indicated numbers of stationary phase L. major promastigotes in 50 µl PBS. Footpad swelling was measured with a metric caliper. For IL-4 neutralization, mice were injected i.p. with 2 mg anti-IL-4, at day -1 and day 0 and subsequently infected with the indicated numbers of stationary phase L. major promastigotes at day 0. Parasite numbers were determined as described (11). Briefly, infected mice were killed 7 wk after infection. Single-cell suspensions were prepared from the spleen and the draining LN, and serial dilutions of these cells were incubated for 2–3 wk in flat-bottom 96-well plates containing complete medium 199. Parasite growth was determined by light microscopy.
Reagents and Abs
LACK recombinant protein was produced and purified as described
(3). LACK (aa 158–173: FSPSLEHPIVVSGSWD) and OVA (aa
323–339: ISQAVHAAHAEINEAG) peptides were purchased from Chiron
Mimotopes (Clayton, Australia). The following mAbs were purchased from
PharMingen (San Diego, CA): 145-2C11, anti-CD3; GK1.5, anti-CD4
(12); 53-6.7, anti-CD8; 16A, CD45RB; IM7,
anti-CD44; MEL-14, anti-CD62L; M1/70, anti-CD11b; RA3-6B2,
anti-B220; HO-13.4, anti-Thy-1.2 (13); M5/114,
anti-I-Ad (14); JES6-5H4 and JES-1A12, anti-IL-2;
11B11 and BVD6-24G2, anti-IL-4; TRFK5 and TRFK4, anti-IL-5;
R4-6A2 and XMG1.2, anti-IFN-. Recombinant mouse IL-2, IL-4,
IL-5, and IFN- were purchased from Genzyme (Cambridge, MA).
Tissue culture medium
DMEM supplemented with 10% heat-inactivated FCS (Corning Costar France, Brumath, France), penicillin (100 U/ml), streptomycin (100 µg/ml), 1 mM sodium pyruvate, L-glutamine (2 mM), and 2-ME (50 µM) was used for all T cell stimulation assays.
Bacterial extracts
The indicated bacteria were lysed in 50 ml of a buffer containing 50 mM Tris-HCl (pH 8), 1 mM DTT, 2 mM EDTA, and one tablet of complete protease inhibitors (Roche Diagnostics, Meylan, France). When indicated, soluble extracts were fractionated using a gel filtration column (Sephacryl S-100 HR, Amersham Pharmacia Biotech, Uppsala, Sweden) run in the same buffer, and individual fractions were collected.
Purification of cells
For purification of CD4+ T cells, LN cells were depleted of B220+, CD11b+, I-Ad+, and CD8+ cells by negative selection using sheep anti-rat-coated Dynabeads as described (10). For adoptive transfer experiments, negatively selected enriched T cells were labeled with FITC-labeled anti-CD4 and PE-conjugated anti-CD62L mAbs and fractionated into CD4+CD62Llow and CD4+CD62Lhigh fractions by two-color sorting on FACStarPlus (Becton Dickinson, San Jose, CA). All populations were >98% pure on reanalysis. For purification of APCs, LN cells were depleted of T cells by cytotoxic elimination using anti-Thy-1.2 mAbs followed by rabbit complement (Low-Tox M, Cedarlane Laboratories, Hornby, Ontario, Canada) and treatment with mitomycin C as described (10).
Flow cytometry
Single-cell suspensions were prepared and stained with optimal concentrations of FITC-labeled and PE-conjugated mAb for 30 min at 4°C in PBS containing 2% FCS. Analysis was performed on a FACScan flow cytometer (Becton Dickinson). Data were collected on 4 x 105 viable cells as determined by forward light scatter and side scatter intensityand were analyzed using Lysis II software (Becton Dickinson).
Ab responses
Abs were measured by ELISA as described (15). Briefly, Immunosorb 96-well plates (Nalge Nunc International, Rochester, NY), were coated with LACK or OVA protein (10 µg/ml) overnight at 4°C. Plates were blocked with 10% FCS-PBS at room temperature for 30 min to prevent non specific binding. Serum was added at serial 3-fold dilutions (starting at 1:10) and incubated overnight at 4°C. HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich Chimie, St. Quentin Fallavier, France) was added for 45 min at room temperature. 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) peroxidase substrate (Sigma-Aldrich Chimie, St. Quentin Fallavier, France) was added, and absorbance was read on an ELISA plate reader (LabSystems, Issy-Les-Moulineaux, France) using a 410-nm pore size filter referenced to 510 nm.
T cell stimulation assays
For stimulation of T cell hybridomas, the indicated Ags were
incubated in round-bottom 96-well plates with 2 x
105 LMR16.2 or LMR17.1D12 T cell hybridomas and
3 x 105 mitomycin C-treated splenocytes
from BALB/c or C57BL/6 mice. Supernatants were harvested 24 h
later and assayed for IL-2 content by ELISA using anti-IL-2
JES6-5H4 and JES-1A12 as capturing and biotinylated detecting mAbs,
respectively. The cytokine content of each sample was determined by
comparison to a standard curve using recombinant mouse IL-2. The
detection limit in all assays was 0.1 U/ml. For anti-CD3
stimulation experiments, ELISA grade 96-well plates were coated with
200 ng affinity-purified anti-CD3 mAbs in 50 mM Tris-HCl, 150 mM
NaCl (pH 9.5) overnight at 4°C. After three washes in PBS, 2 x
106 splenocytes from SPF or GF mice were added to
each well. Supernatants were harvested 48 h later and analyzed for
IFN- and IL-4 contents by ELISA. For T cell proliferation assays,
2 x 105 CD4+ T cells
from the TCR-LACK C° or DO.11.10 transgenic mice were
incubated for 72 h with the indicated numbers of mitomycin
C-treated APCs in round-bottom 96-well plates. Cells were pulsed with 1
µCi [3H]thymidine during the last 18 h
of culture, and plates were harvested and counted on a Betaplate Reader
(Amersham Pharmacia Biotech, Uppsala, Sweden). For other T cell assays,
106 CD4+ T cells from
immunized or infected mice were incubated in flat-bottom 96-well plates
with 106 mitomycin C-treated splenocytes and the
indicated concentrations of Ags. Supernatants were harvested 48 or
72 h later and analyzed for IFN-, IL-4, and IL-5 contents by
ELISA.
Competitive RT-PCR
Total RNA was extracted using RNAeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Reverse transcription was performed using murine Moloney leukemia virus RT (Life Technologies, Cergy Pontoise, France) and oligo(dT) primers (Amersham Pharmacia Biotech). Semiquantitative PCR was performed as described (16). In brief, cDNA was first assayed for levels of the constitutively expressed gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT), by incubating different concentrations of the polycompetitor construct and determining the ratio of competitor to wild-type band intensity after amplification with HPRT-specific primers. The adjusted volumes of cDNA were used to measure IL-4 or IL-10 mRNA levels, using IL-4- or IL-10-specific primers. After separation of the PCR products by electrophoresis in agarose gel, the ratio of the relative concentration of IL-4 or IL-10 mRNA to the relative concentration of HPRT mRNA was calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Naive and memory/effector T lymphocytes express different levels
of surface molecules such as CD62L, CD45RB, and CD44 (17).
Thus, in contrast to most naive T cells, which express high levels of
CD62L and CD45RB and low levels of CD44, memory/effector T lymphocytes
express low levels of CD62L and CD45RB and high levels of CD44. To
analyze the phenotype of LACK-specific T cells in uninfected mice,
CD4+ T cells from BALB/c mice were sorted on the
basis of CD62L expression and adoptively transferred into BALB/c nude
recipients. Mice were immunized with LACK or OVA, and Ag-specific Ab
responses were monitored by ELISA. As expected, OVA-specific Abs were
detectable in the sera of the mice that had been injected with
CD62Lhigh or unseparated
CD4+ cells, but not in the sera of the recipients
reconstituted with CD62Llow cells (Fig. 1
A). In contrast,
LACK-specific Ig were detected in the sera of all reconstituted animals
including those that had received the memory/effector
CD62Llow CD4+ cells (Fig. 1B). Thus, whereas all OVA-specific T cells expressed high
levels of CD62L, LACK-specific T cells were found in both the
CD62Llow and CD62Lhigh
populations, suggesting that at least some of them had been stimulated
by cross-reactive Ags before infection with the parasite.
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° transgenic mice in which all T
cells express a single receptor recognizing a LACK-derived
immunodominant peptide bound to I-Ad molecules
(9). In contrast to what was observed in other monoclonal
TCR transgenic mice (18, 19), a majority of
CD4+ T cells in TCR-LACK C° transgenic
mice were large CD62Llow
CD44high CD45RBlow cells
(Fig. 2). Interestingly, the proportion
of transgenic T cells exhibiting this large
CD62lowCD44highCD45RBlow
phenotype was higher in the mesenteric LN than in other secondary
lymphoid organs including the spleen (Fig. 2) or the popliteal and
inguinal LN (data not shown).
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The higher proportion of large
CD62lowCD44highCD4+
T cells found in the mesenteric LN of TCR-LACK C° mice could
result from the preferential trafficking of these cells to the
mesenteric LN. Alternatively, LACK-specific T cells could be primed in
the mesenteric LN, possibly by exposure to cross-reactive Ags present
in the digestive tract. To test this latter hypothesis,
CD4+ T cells from TCR-LACK C°
transgenic mice were incubated with APCs purified from the secondary
lymphoid organs of BALB/c mice. In contrast to APCs isolated from the
spleen or the popliteal LN, APCs from the mesenteric LN induced the
proliferation of LACK-specific CD4+ transgenic T
cells (Fig. 3). This proliferation was
dependent on the number of APCs and was blocked by
anti-I-Ad mAbs (data not shown). In contrast,
OVA-specific CD4+ T cells from DO11.10 transgenic
mice did not proliferate when incubated with mesenteric LN APCs. Thus,
APCs from the mesenteric LN of naive BALB/c mice are loaded with
peptides that can stimulate LACK-specific T cells.
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Because Ags from the intestinal flora are the most abundant and
diverse Ags to which T cells are exposed in SPF mice, we sought to
determine whether bacteria isolated from the gut of BALB/c mice
contained Ags that could stimulate LACK-specific T cells. To address
this issue, we incubated five different LACK-specific T cell hybridomas
with crude extracts from various aerobic and anaerobic bacteria.
Extracts from Escherichia coli and Enterococcus
faecalis, but not those from Proteus mirabilis and
Clostridium perfringens induced IL-2 secretion by LMR16.2
and LMR17.1D12 T cell hybridomas in a dose-dependent manner (Fig. 4A and data not shown). In
contrast, other LACK-specific T cell hybridomas, i.e., LMR8.1, LMR8.3,
and LMR4.1, did not secrete IL-2 in response to these extracts (data
not shown).
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B). As an attempt to identify microbial proteins that could
stimulate LACK-specific T cell hybridomas, soluble extracts from
E. faecalis were fractionated on a gel filtration column,
and individual fractions were tested for protein content and for the
ability to stimulate LMR16.2 T cell hybridomas. Stimulatory activity
was found in two groups of four consecutive fractions corresponding to
proteins of 30–35 kDa and high molecular mass (>90 kDa), respectively
(Fig. 4C).
Although soluble extracts from E. faecalis and
Escherichia coli stimulated IL-2 secretion from two
different LACK-specific T cell hybridomas, we sought to determine
whether cross-reactivity between LACK and bacterial Ags could also be
demonstrated using polyclonal populations of T cells. To this end,
BALB/c mice were immunized with bacterial extracts from E.
faecalis, and T cells from their LN were incubated with LACK or
soluble bacterial proteins. We found that T cells from nonimmunized
BALB/c mice did not secrete any detectable amount of IFN- or IL-4
when incubated with either LACK or E. faecalis extracts
(Fig. 4D). In contrast, T cells from mice immunized with
E. faecalis extracts secreted small but detectable amounts
of IFN- in response to LACK peptide in vitro. As expected, T cells
from immunized mice secreted high amounts of IFN- in response to
soluble E. faecalis Ags (Fig. 4D).
Decontamination of the digestive tract prevents both the development of memory/effector LACK-specific CD4+ T cells and the early burst of IL-4 mRNA induced by L. major
If exposure to bacterial Ags was responsible for the
memory/effector phenotype expressed by LACK-specific T cells before
infection, these cells should exhibit a naive phenotype in mice in
which the digestive tract has been decontaminated with antibiotics.
Indeed, treatment of TCR-LACK C° transgenic mice with
antibiotics starting at 2 wk of age resulted in a dramatic reduction in
the proportion of CD4+ T cells expressing low
levels of CD62L (Fig. 5A).
Moreover, the early accumulation of IL-4 mRNA which was detected
16 h after infection in the draining LN of SPF BALB/c mice was not
observed in either antibiotic-treated SPF or GF BALB/c mice (Fig. 5B, left). Similarly, no increase in IL-4 mRNA
levels was observed in the spleens of antibiotic-treated SPF or GF mice
following i.v. inoculation of L. major promastigotes (Fig. 5B, right). This latter phenomenon was not due to
a defect in the ability of CD4+ T cells from GF
mice to secrete IL-4. Indeed, CD4+ T cells from
GF and SPF BALB/c mice secreted similar amounts of IL-4 and IFN-
when stimulated in vitro with anti-CD3 mAbs (Fig. 5C).
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). In striking contrast,
the early accumulation of IL-10 mRNA induced by the parasite was
dramatically reduced in both GF BALB/c mice and SPF IE-LACK transgenic
mice (Fig. 6).
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Because GF BALB/c mice did not exhibit a parasite-induced IL-4
burst, we sought to determine whether these mice were resistant to
L. major. To this aim, GF and SPF BALB/c mice were infected
with L. major promastigotes. Unexpectedly, we found that
both GF and SPF mice developed progressive lesions (Fig. 7A) and exhibited high numbers
of parasites in their spleen and LN (Fig. 7B). Indeed, the
LN and the spleens of GF mice contained 2.5- and 12-fold more
parasites, respectively, than those of SPF animals, suggesting that GF
mice were even more susceptible to L. major than SPF
mice.
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B).
In striking contrast, anti-IL-4-treated GF mice developed lesions
that were larger than those exhibited by untreated GF or SPF animals.
Although the lesions developed by anti-IL-4-treated GF mice did not
increase in size at later time points, they remained larger than those
exhibited by anti-IL-4-treated SPF mice. In agreement with the
latter result, the LN of anti-IL-4-treated GF mice contained
12-fold more parasites than those from anti-IL-4-treated SPF mice.
Likewise, whereas parasites were not detectable in the spleens of
anti-IL-4-treated SPF mice, the spleens of anti-IL-4-treated GF
mice contained 3- to 4-fold more parasites than those from untreated
SPF animals (Fig. 7B). Thus, whereas the early
neutralization of IL-4 allowed SPF mice to control the replication of
the parasite and to resolve the infection, anti-IL-4-treated GF
mice remained highly susceptible to L. major. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because molecular probes to visualize the LACK-specific T cells were
not available, we used an adoptive transfer system to analyze the
surface phenotype of these cells in BALB/c mice. In contrast to
OVA-specific T cells which were only found within the
CD4+CD62Lhigh population,
LACK-specific T cells were found in both the
CD4+CD62Lhigh and
CD4+CD62Llow subsets.
Although this result suggested that at least some LACK-specific T cells
had been exposed to an Ag, we did not know which proportion of these
cells expressed a naive or a memory/effector phenotype, respectively.
For this reason, we have analyzed the surface phenotype of
CD4+ T cells in TCR-LACK C° transgenic
mice in which all CD4+ T cells expressed a single
TCR recognizing a LACK-derived peptide bound to
I-Ad molecules (9). Although the
proportion of CD4+ cells expressing a
memory/effector phenotype were variable from one mouse to another, the
spleen and the LN of these mice always contained a large fraction of
CD4+ T cells expressing low levels of CD62L and
CD45RB and high levels of CD44. Thus, in TCR-LACK C°
transgenic mice, 40–50% and 60–70% of splenic
CD4+ T cells expressed low levels of CD62L and
high levels of CD44, respectively. Moreover, because late
memory/effector cells can sometimes revert to a naive phenotype, it is
possible that some of the LACK-specific T cells which expressed high
levels of CD62L and/or low levels of CD44 were not true naive cells,
but had reverted to this phenotype after a first antigenic
stimulation.
Several piece of evidence suggests that the memory/effector phenotype
expressed by LACK-specific T cells results from their priming by
cross-reactive microbial Ags derived from the gut flora. First, the
fractions of CD4+ T cells expressing low levels
of CD62L or high levels of CD44 were higher in the mesenteric LN of
TCR-LACK C° transgenic mice than in any other secondary
lymphoid organs. In mesenteric LN, most LACK-specific T cells expressed
a large phenotype typical of actively dividing cells. The latter
phenomenon was not observed in either BALB/c mice or DO.11.10 TCR
transgenic mice in which a large number of T cells reacted to an OVA
peptide bound to I-Ad molecules. Secondly, APCs
from the mesenteric LN of BALB/c mice, but not those from the spleen or
the popliteal LN, were able to stimulate the proliferation of
transgenic LACK-specific T cells in the absence of exogenously added
Ags. In contrast, no proliferation was observed when OVA-specific
CD4+ T cells from DO.11.10 TCR transgenic mice
were incubated with mesenteric LN APCs. In this respect, it is
noteworthy that the TCR transgenic mice that we have used in this study
have been maintained as heterozygotes by successive crosses to the same
genetic stock of BALB/c mice. Thus, the fact that T cells from TCR-LACK
C°/° mice proliferated in response to
BALB/c APCs was not due to subtle mismatches in minor
histocompatibility Ags selectively expressed by mesenteric and not
popliteal APCs. Thirdly, cells from two of five different LACK-specific
T cell hybridomas secreted IL-2 when incubated with crude or partially
purified soluble extracts from Escherichia coli or E.
faecalis. Although the amounts of IL-2 secreted in response to
bacterial Ags were small compared with those secreted in response to
recombinant LACK protein, IL-2 secretion was observed only in the
presence of syngeneic APCs and was blocked by
anti-I-Ad Abs. Moreover, only two of five
LACK-specific T cell hybridomas secreted IL-2 in response to bacterial
extracts, ruling out the possibility that this phenomenon was the
result from the activation of APCs by bacterial products such as LPS or
lipoteichoic acid. Although we were not able to detect a T cell
response against either LACK or E. faecalis soluble proteins
in naive BALB/c mice, this could easily be explained if the frequency
of the LACK-specific T cells was not high enough. In contrast, T cells
from mice that had been immunized with E. faecalis extracts
responded to LACK in vitro. Although it may be surprising that IFN-
but not IL-4 was detected in this assay, it could be that s.c.
immunization with bacterial extracts favored the development of
LACK-specific Th1 cells, as opposed to natural priming in GALTs. Thus,
experiments performed with T cell hybridomas, T cells from TCR
transgenic mice, or polyclonal populations of T cells pointed to
cross-reactivity between LACK and bacterial soluble proteins. Fourthly,
the fraction of LACK-specific T cells expressing a memory/effector
phenotype was reduced in TCR-LACK C° in which the digestive
tract had been decontaminated with antibiotics. Lastly, we have
recently shown that a subset of CD4+ T cells from
TCR-LACK C° mice, but not from DO.11.10 scid mice,
adoptively transferred colitis when injected into C.B.-17.
scid mice (unpublished results).
Although most LACK-specific T cells express V8 and V4,
LACK-specific TCR can use very distinct CDR3 regions (our unpublished
results). Thus, the T cell response directed to the LACK immunodominant
peptide is highly polyclonal. In this respect, it would be interesting
to determine which proportion of the LACK-specific T cells react to gut
flora Ags. Although we do not have the answer to this question, it is
noteworthy that only two of five LACK-specific T cell hybridomas
secreted IL-2 in response to E. faecalis extracts. Thus,
although our results suggest that the parasite-induced IL-4 burst
depends on the priming of LACK-specific T cells by microbial Ags, it is
likely that only a fraction of the LACK-specific T cells are
cross-reactive to these Ags and eventually contribute to the rapid
production of IL-4 induced by L. major. Experiments are in
progress to identify structural features of LACK-specific TCR which
would allow them to respond to bacterial extracts.
In an attempt to identify the gut flora Ags which were responsible for the priming of the LACK-specific T cells, we have searched Escherichia coli open reading frames for sequences exhibiting similarities to the LACK immunodominant peptide (SLEHPIVVSGSW) using the BLAST program (22). We identified 61 sequences and synthesized the corresponding peptides. Although one of these peptides (VLQHPLVTTAFS), which was derived from the Escherichia coli lit gene product, stimulated LMR16.2 T cell hybridomas in vitro, we do not know whether this peptide was actually presented to T cells in vivo and, even if this was the case, how many other microbial peptides were able to stimulate LACK-specific T cells. Indeed, several fractions from E. faecalis extracts were capable of stimulating the LACK-specific hybridomas LMR16.2 and LMR17.1D12, suggesting that these bacteria contained more than one LACK mimicry Ag. Also, additional experiments showed that only high concentrations of the lit-derived peptide were able to stimulate LMR16.2 T cell hybridomas and that this peptide bound poorly to I-Ad molecules. Thus, the bacterial Ags which were responsible for the priming of LACK-specific T cells in vivo remain to be identified.
LACK-specific CD4+ T cells are rapidly activated to secrete IL-4 in BALB/c mice infected with L. major. Indeed, as early as 16 h after infection, a burst of IL-4 mRNA could be detected in the draining LN of BALB/c mice, but not in animals that had been made tolerant to LACK by immunological manipulations or transgenic expression of this Ag in the thymus (23). Here, we found that decontamination of the digestive tract of SPF mice with antibiotics caused a dramatic reduction in the early burst of IL-4 induced by the parasite. Likewise, no early IL-4 burst was observed in GF mice. As expected for T cells that have been primed in GALTs (20), we found that L. major induced the rapid accumulation of IL-10 mRNA in SPF BALB/c mice. However, this IL-10 burst was dramatically reduced in both BALB/c GF mice and IE-LACK transgenic SPF mice that were tolerant to LACK as the result of the expression of this Ag in the thymus. Thus, our results suggest that the priming of LACK-specific T cells by microbial Ags from the gut flora is critical in determining their ability to rapidly produce IL-4 and IL-10 mRNA in response to L. major.
Many studies have suggested that there is a causal relationship between
the early accumulation of IL-4 transcripts in the draining LN of BALB/c
mice and the development of a counterprotective Th2-like immune
response against L. major. Thus, the early burst of IL-4
mRNA induced by the parasite in susceptible BALB/c mice was not
observed in resistant strains including C57BL/6, C3H/He, and CBA/Ca. No
accumulation of IL-4 mRNA was found in BALB/c mice that were made
resistant by the early administration of IFN- or IL-12
(24). In contrast, treatment of resistant C57BL/6 mice
with neutralizing anti-IFN- mAbs induced both the early
accumulation of IL-4 mRNA and susceptibility (24). Despite
these data showing that IL-4 is critical for susceptibility, some other
studies have suggested that other yet unidentified factors may be
capable of causing susceptibility to L. major in the absence
of IL-4 or IL-4 signaling (25, 26). At least two
hypotheses could account for these apparently conflicting data. Thus,
it is possible that the very rapid accumulation of IL-4 transcripts
which is induced by the parasite in BALB/c mice would not be required
for susceptibility as was previously thought. In this case, priming of
LACK-specific T cells by L. major cross-reactive intestinal
microbial Ags would not be causally related to susceptibility but might
be an innocent bystander effect resulting from a possible high
frequency of LACK-specific CD4+ T cells. We think
this hypothesis is unlikely for two reasons. First, it was recently
found that administration of IL-4 to BALB/c mice that were depleted
from LACK-specific T cells was sufficient to instruct Th2 cell
development and to restore susceptibility to L. major
(27). Secondly, we have tried to determine the proportion
of CD4+ T cells reacting to LACK in BALB/c mice
before and 1 day after infection with L. major. Although
LACK-specific T cells could not be detected in naive mice, the
frequency of these cells among CD4+ T was below 1
of 5 x 104 in infected animals as
determined by an IL-4 ELISPOT assay. Thus, although we cannot rule out
that the number of LACK-specific T cells would be underestimated using
this assay, this frequency does not seem to be different from those
reported for other Ag-specific CD4+ T cells
(28). Alternatively, the lack of an intestinal flora in
antibiotic-treated SPF and GF mice may result in complex physiological
alterations that could impair their ability to control the infection.
In agreement with this latter hypothesis and in contrast to what was
observed in SPF BALB/c mice, we found that anti-IL-4-treated GF
BALB/c mice did not control the replication of the parasite and
remained susceptible to L. major infection. Likewise, a
recent study has shown that GF Swiss mice developed progressive lesions
when infected with L. major, whereas SPF Swiss mice only
make small lesions and eventually heal (29). Lastly,
macrophages derived from the spleen of GF mice expressed reduced
amounts of MHC class II molecules (30) and exhibited
impaired ability to secrete IL-1, IL-6, and IL-12
(31, 32, 33). Thus, although T cells from GF mice differ from
those from SPF mice because they have not been exposed to gut flora
Ags, GF mice exhibit many other defects that could explain why they
remained susceptible to L. major despite the lack of the
parasite-induced IL-4 burst.
It would be interesting to know whether bacteria-cross-reactive LACK-specific T cells also develop in mice from resistant strains. Although we have not directly addressed this issue, we recently found that mice from the resistant B10.D2 strain exhibited an early burst of IL-4 mRNA when infected with L. major (23). Moreover, similarly to what we have observed in BALB/c mice, the early production of IL-4 was completely abolished when B10.D2 mice were made tolerant to LACK (23). Although we do not know whether antibiotic-treated or GF B10.D2 mice would exhibit a burst of IL-4 mRNA, our data suggest that priming of the LACK-specific T cells by cross-reactive bacterial Ags may not be restricted to susceptible BALB/c mice, but rather to mice of the d haplotype.
Because of their location, it is not surprising that bacteria from the intestinal flora play a critical role in both the development and the function of the mucosal immune system. Thus, as compared with SPF mice or GF mice colonized with single or multiple species of bacteria, GF mice exhibit smaller Peyer’s patches, reduced numbers of IgA-secreting plasmacytes (34) and intraepithelial lymphocytes (35, 36), and are partially or totally resistant to the induction of oral tolerance to some Ags (37, 38, 39, 40). In contrast to this large body of literature, very few data are available on the role of the intestinal flora on systemic Ag-specific immune responses (41). In this respect, the results reported here are the first to demonstrate that priming of helper T cells by cross-reactive bacterial proteins in GALTs may be critical in determining their ability to rapidly respond to their cognate Ag in peripheral lymphoid organs. Thus, a key parameter of the immune response to a cutaneous parasite may be a consequence of T cell cross-priming by microbial Ags from the indigenous intestinal flora.
Although several studies have demonstrated that the mucosal immune system of healthy individuals is unresponsive to bacterial Ags from the gut flora, the molecular and cellular mechanisms that are responsible for the establishment and/or the maintenance of tolerance are still poorly understood (42, 43, 44). Thus, it is not known when and where bacteria-specific T cells encounter these Ags for the first time and how these T cells behave when they have been primed. Although more studies are needed to generalize these findings, our results suggest that bacteria-specific CD4+ T cells that have been primed in GALTs are not deleted but recirculate through the spleen and the LN, where they can rapidly produce IL-4 and IL-10 when stimulated by cross-reactive Ags. Thus, tracking the fate of the LACK-specific T cells in BALB/c mice may provide important information about the regulation of cell-mediated immune responses to gut Ags in both physiological and pathological conditions.
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Acknowledgments |
|---|
° transgenic mice, C.
Morrissey for their help with antibiotic treatment, T. Champion
for peptide synthesis, J. Bigay and S. Beraud for their
help with protein purification, C. Moreau and F. Powrie for
helpful discussions, and R. Coffman for his support. |
Footnotes |
|---|
2 Current address: DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304-1104.
3 Current address: Microbiology Department, University of Minnesota, 312 Church Street, SE, Minneapolis, MN 55455.
4 Address correspondence and reprint requests to Dr. Nicolas Glaichenhaus, Center National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 Route des Lucioles, 06560 Valbonne, France.
5 Abbreviations used in this paper: LACK, Leishmania homologue of mammalian RACK1; LN, lymph node; GALTs, gut-associated lymphoid tissues; GF, germfree; SPF, specific pathogen free; HPRT, hypoxanthine-guanine phosphoribosyltransferase; CD62L, CD62 ligand.
Received for publication August 2, 1999. Accepted for publication August 28, 2000.
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) or purified
CD62Lhigh (
) or CD62Llow (•)
CD4+ T cells from the mesenteric LN of naive BALB/c mice
were prepared, and 1.5 x 106 cells were injected i.v.
into BALB/c nude recipients. Control animals were not injected (
).
After 2 wk, mice were immunized i.p. with 50 µg of OVA
(A) or LACK (B) in PBS. Mice were boosted
2 wk after the first immunization and analyzed 5 days later for the
presence of serum IgG OVA (A)- or LACK
(B)-specific Abs by ELISA.
) or popliteal (
), or not
infected (
