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koberneInstitut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is an essential component of the early Listeria
monocytogenes-specific immune response, and is also an
important regulator of Ag processing and presentation. Ag presentation
is required for the induction and also the effector function of
antimicrobial T cells. To evaluate the effect of IFN- on bacterial
Ag presentation in vivo, macrophages and dendritic cells were separated
from L. monocytogenes-infected tissues and analyzed with
peptide-specific CD4 and CD8 T cell lines in a sensitive ELISPOT-based
ex vivo Ag presentation assay. The comparison of professional APCs
isolated from infected IFN--deficient and wild-type mice revealed
different peptide presentation patterns of L.
monocytogenes-derived CD8 T cell epitopes, while the
presentation pattern of CD4 T cell epitopes remained unchanged. The
further in vitro analysis of the generation of CD8 T cell epitopes
revealed a peptide-specific effect of IFN- on MHC class I-restricted
Ag presentation. These results show that despite this modulation of the
Ag presentation pattern of CD8 T cell epitopes, IFN- is not
generally required for the MHC class I- and MHC class II-restricted
presentation of L. monocytogenes-derived antigenic
peptides by professional APCs in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is secreted by activated
CD8 T cells, the Th1 subset of CD4 T cells, and by NK cells. The
functions of IFN- include the regulation of the Th cell response,
the stimulation of the bactericidal activity of phagocytes, and the
regulation of Ag presentation through class I and class II MHC
molecules (1).
The resistance against the facultatively intracellular bacterium
Listeria monocytogenes is IFN- dependent. IFN- is
strongly secreted during the early T cell-independent phase of a
primary L. monocytogenes infection (2). NK
cells represent an important source of IFN- during the early phase
of the murine L. monocytogenes infection. Mice with a
targeted disruption of the genes coding for IFN-
(GKO)3
(3) or the IFN- receptor
(RKO) (4) die after infection with a relatively low dose
of L. monocytogenes. Generally, it is believed that the most
important function of IFN- during L. monocytogenes
infection is the stimulation of the killing of bacteria in infected
macrophages (5, 6, 7). For the T cell-dependent antilisterial
immunity, IFN- is not essential (3). Recently, the
analysis of listeriolysin O (LLO)91–99- and
p60217–225-specific CD8 T cells in wild-type
(wt) and GKO mice revealed that also the relative immunodominance of
antilisterial CD8 T cell populations is IFN- dependent
(8).
IFN- is a potent regulator of Ag processing and presentation.
Multiple essential components of the cytosolic Ag presentation pathway
are subject to IFN- regulation, e.g., the expression of TAP
(9), the expression of proteasome subunits
(10), and the expression of the MHC class I heavy chain
and 
2-microglobulin genes (11).
The cytosolic degradation of Ag is thought to be a function of the
proteasome, in which three subunits, LMP2, LMP7, and multicatalytic
endopeptidase complex-like 1, and also the PA 28 regulator are induced
by IFN- (reviewed in Ref. 10). IFN- regulation of
the endosomal Ag presentation pathway includes the expression of
endosomal proteases (12, 13) and of MHC class II molecules
(14).
To evaluate the effect of IFN- on microbial Ag presentation in vivo,
macrophages and dendritic cells (DC) were separated from L.
monocytogenes-infected spleens and analyzed with peptide-specific
CD4 and CD8 T cell lines using the recently developed, sensitive
ELISPOT-based ex vivo Ag presentation assay (15). Our data
show that despite IFN- modulation of the Ag presentation pattern of
CD8 T cell epitopes, IFN- is not necessary for the efficient MHC
class I- and MHC class II-restricted presentation of bacterial T cell
epitopes by professional APC in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/cOlaHsd (H-2d), C57BL/6
(H-2b), and SV129 (H-2b)
mice were purchased (Harlan-Winkelmann, Borchen, Germany), kept under
conventional conditions, and used at 8–10 wk of age. BALB/c mice with
a targeted mutation of the gene coding for IFN-
(C.129S7(B6)-Ifngtm1Ts (16))
and 129-Ifngrtm1 mice (4)
with a targeted mutation of the gene coding for the IFN- receptor
were obtained from The Jackson Laboratory (Bar Harbor, ME) and were
bred under conventional conditions. Mice were infected with L.
monocytogenes serovar 1/2a EGD in 0.2 ml PBS i.v., as indicated.
Bacteria used for infection were in the logarithmic growth phase. The
bacterial concentration was estimated from the
OD600.
CD4 and CD8 T cell lines
CD8 T cell lines specific for p60217–225,
p60449–457, p60476–484,
and LLO91–99 were derived from spleens of
L. monocytogenes-infected BALB/c mice. A
Ld-restricted murine CMV
pp89168–176-specific CD8 T cell line
(17) was kindly provided by R. Holtappels (University of
Mainz, Mainz, Germany). All CD8 T cell lines were propagated by
repeated restimulation with P815 cells transfected with the human B7.1
gene (P815/B7) (18) in the presence of the appropriate
synthetic peptide in medium supplemented with IL-2, as described
previously (19). CD4 T cell lines specific for
LLO190–201, LLO318–329,
LLO253–264, and
p60177–188 were established from spleens 14 days
after i.v. infection of C57BL/6 mice with 1 x
103 CFU L. monocytogenes. CD4 T cell
lines were repeatedly restimulated with mitomycin C-inactivated
splenocytes as APC in the presence of 10-6 M
peptide. The T cell culture medium was 
modification of Eagle’s
medium (Invitrogen, Karlsruhe, Germany) supplemented with glutamine,
penicillin, streptomycin, 10% FCS, 100 U/ml murine rIL-2 (R&D Systems,
Wiesbaden Germany), and 2 x 10-5 M 2-ME.
Immunomagnetic isolation and cytofluorometric analysis of macrophages and DC
Cells expressing CD11b or CD11c were isolated by immunomagnetic cell sorting from spleens of L. monocytogenes-infected mice. Cells were selected with paramagnetic microbeads conjugated to monoclonal hamster anti-mouse-CD11b (clone M1/70.15.11.5; Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-mouse CD11c Abs (clone N418; Miltenyi Biotec), respectively. Spleens were removed 48 h after i.v. infection of mice. Spleens were injected with 500 µl of a 1 mg/ml solution of collagenase D (Roche Diagnostics, Mannheim, Germany) in HBSS. Subsequently, spleens were cut in small pieces and incubated 30 min at 37°C, 5% CO2 in the collagenase D buffer. Cells were collected by centrifugation and separated twice on MS+ positive selection columns (Miltenyi Biotec) following the standard positive selection protocol provided by the manufacturer. For each experiment, spleens from three mice were pooled. At the end of the positive selection procedure, between 0.5 x 106 and 2 x 106 positive cells were obtained per 1 x 108 spleen cells. Aliquots of the selected cells were stained with FITC-labeled rat anti-mouse CD11b IgG2b mAb (clone M1/70; BD PharMingen, San Diego, CA), rat Ig2b isotype control mAb (A95-1; BD PharMingen), hamster anti-mouse CD11c IgG mAb (clone HL3; BD PharMingen), and hamster IgG isotype control mAb (clone G235-2356; BD PharMingen), respectively, and subjected to FACS analysis. Cells selected with anti-CD11c microbeads were generally >80% pure DC. A differential cell count revealed that macrophage-like mononuclear cells selected by anti-CD11b microbeads were contaminated by approximately 30% polymorphonuclear granulocytes. For the analysis of MHC class I and II expression, isolated DC were stained with FITC-labeled hamster anti-mouse CD11c IgG mAb and PE-labeled rat anti-mouse I-A/I-E IgG2b mAb (clone M5/114.15.2; BD PharMingen) or with unlabeled rat anti-mouse MHC class I IgG2a mAb (clone ER-HR 52; Bachem/Peninsula Laboratories, San Carlos, CA), followed by PE-labeled goat anti-rat IgG mAb (Southern Biotechnology Associates, Birmingham, AL), respectively.
Isolation of endogenously processed peptides
Peptide extraction from infected cells and organs was performed
as described previously (15). Briefly, spleens were
removed 48 h after i.v. infection with 1 x
106 (BALB/c wt mice) or 1 x
104 (GKO mice) CFU L. monocytogenes.
Trifluoroacetic acid was added to organ homogenates to achieve a pH of
2. The lysis solution was supplemented with Complete proteinase
inhibitor and pepstatin (both Roche Diagnostics). Afterward, extracts
were sonicated and centrifuged for 1 h at 50,000 x
g. Supernatants were removed and passed through a Sephadex
G-25 (Amersham Pharmacia, Freiburg, Germany) column. Low m.w. fractions
were collected and passed through a SepPak C18
reversed-phase solid-phase extraction unit (Waters, Eschborn, Germany).
Bound hydrophobic material was eluted, concentrated by vacuum
centrifugation, and further fractionated by HPLC on a reversed-phase
C18 column (
Pak
C18-300A, 3.9 x 300 mm; Waters): 1 ml
peptide extract was loaded and eluted with a flow rate of 1 ml/min on a
linear acetonitrile gradient. Solution A, 0.1% trifluoroacetic acid;
solution B, 70% acetonitrile, 0.09% trifluoroacetic acid. Gradient:
0- to 5-min 0% B, 5- to 55-min linear increase to 50% B; 55- to
63-min linear increase to 100% B; 63- to 66-min 100% B; 66- to 74-min
linear decrease to 0% B. One-minute fractions were collected and
stored at -70°C.
Isolation of naturally processed peptides from in vitro infected
macrophage-like P388D1 (P388) cells was performed
similarly. IFN- pretreatment of P388 cells was performed for 24
h with 100 U/ml murine rIFN- (R&D Systems). Approximately 1 x
108 adherent P388 cells were infected with
L. monocytogenes at a multiplicity of infection of 10. After
1 h at 37°C, cells were washed once, and the medium was
exchanged with medium containing 5 µg/ml gentamicin. After further
5-h incubation at 37°C, cells were washed twice with ice-cold PBS and
were subsequently harvested with a cell scraper. Cell pellets were
disrupted, sonicated, and lysed 30 min in 2 ml 0.5% trifluoroacetic
acid supplemented with proteinase inhibitors, as described above.
Subsequently, cell lysates were centrifuged for 30 min at 20,000
x g, and supernatants were further purified by
ultrafiltration using Microsep (Pall-Gelman, Dreieich, Germany)
ultrafiltration units with 10-kDa cutoff. The low m.w. fraction was
further fractionated by HPLC, as described above.
Quantification of endogenously processed peptides
The precise amount of antigenic peptides in HPLC fractions was
determined as described previously (15). Fractions were
tested in a standard chromium release assay with
51Cr-labeled P815 cells as APC. The peptide
concentration of the fractions was calculated by linear interpolation
from lysis data obtained with a synthetic peptide standard. Recovery
from spleen extracts was approximately 20% for
p60217–225 and
p60476–484, 10% for
p60449–457, and 60% for
LLO91–99. Recovery from cell extracts was
20% for all three p60 peptides and 70% for
LLO91–99. In the calculation of the total number
of peptides per organ or per cell, the different recovery rates were
counted.
ELISPOT-based Ag presentation assay
Ag presentation by in vivo infected cells was assessed with an
ELISPOT-based Ag presentation assay, as described previously
(15). This assay applies the basic principle of the
ELISPOT assay for the detection of Ag presentation by target cells that
acquired Ag in vivo. Splenocytes were used as APC after passing through
nylon gaze (80 mesh) and RBC lysis. Alternatively, macrophages and DC
isolated from infected spleens were used as APC. The ELISPOT assay was
also used to detect antigenic material contained in HPLC fractions from
peptide extracts. P815 cells were used as APC and were loaded with
peptides, as described above. The setting of the assay was similar for
all APC types. All APC were tested in the presence of 10 µg/ml
gentamicin and 20 µg/ml tetracycline. In round-bottom 96-well
microtiter plates, 3 x 104 peptide-specific
CD8 T cells were added per well to graded numbers of APC in a final
volume of 150 µl. After 5-h preincubation, cells were resuspended,
and 100 µl cell suspension was transferred to rat anti-mouse
IFN- mAb-coated (RMMG-1; Biosource, Camarilla, CA) nylon
membrane-backed 96-well microtiter plates (Nunc, Wiesbaden, Germany)
and incubated overnight. ELISPOT plates were developed with
biotin-labeled rat anti-mouse IFN- mAb (clone XMG1.2; BD
Biosciences, Heidelberg, Germany), HRP streptavidin conjugate (Dianova,
Hamburg, Germany), and aminoethylcarbazole dye solution.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on the generation of MHC class I-restricted
antigenic peptides in vivo
To test the effect of IFN- on the presentation of L.
monocytogenes-derived antigenic peptides, a sensitive
ELISPOT-based ex vivo Ag presentation assay was used (15).
The ELISPOT-based Ag presentation assay is a qualitative way to assess
Ag presentation by ex vivo isolated APCs. It is a highly sensitive
alternative to other commonly used Ag presentation assays based on the
quantification of cytokines, proliferation of responder cells, or
killing of target cells. As other Ag presentation assays, the
ELISPOT-based assay also is used for the direct comparison of different
APC. GKO and wt mice were infected i.v. with 5 x
104 CFU L. monocytogenes, and 48
h postinfection (p.i.) splenocytes were tested with CD8 T cell lines
specific for different CD8 T cell epitopes of L.
monocytogenes. T cell activation was measured in an
IFN--specific ELISPOT assay. Fig. 1
shows the peptide presentation patterns of wt and GKO splenocytes. The
strength of T cell activation, but not the activation pattern
depended on the number of spleen cells present. On splenocytes of both
wt and GKO mice, p60217–225 and
LLO91–99 were the strongest peptides detected.
In contrast to wt splenocytes that presented
LLO91–99 stronger than
p60217–225, the LLO91–99
epitope was presented weaker than p60217–225 on
GKO splenocytes. Presentation of peptides
p60449–457 and p60476–484
was also detected. While the presentation of
p60476–484 remained constant in wt and GKO mice,
the presentation of p60449–457 was stronger on
GKO splenocytes compared with wt splenocytes. As a control,
pp89168–176-specific CD8 T cells also were
included that were not activated by L.
monocytogenes-infected APC.
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on the generation of
L. monocytogenes-derived CD8 T cell epitopes, naturally
processed antigenic peptides were extracted from spleens of infected wt
and GKO mice. BALB/c wt and GKO mice were infected i.v. with 1 x
106 and 1 x 104 CFU
L. monocytogenes, respectively. These infectious doses were
chosen to give similar bacterial loads in wt and GKO spleens 48 h
after infection. Spleens were removed, and naturally processed
antigenic peptides were extracted, separated by HPLC, and quantified.
Fig. 2A shows the test of HPLC
fractions from organ extracts with peptide-specific CD8 T cell lines in
a 51Cr release assay. HPLC fractions 30/31 and
33/34 reacted with LLO91–99- and
p60217–225-specific CD8 T cell lines,
respectively. Peptides p60449–457 and
p60476–484 were not detected by CD8 T cells of
corresponding specificity (data not shown). The amount of
p60217–225 and LLO91–99
peptide present in infected spleens was quantified by linear
interpolation from an external peptide standard. Fig. 2B
shows the number of extracted antigenic peptides per spleen for wt and
GKO mice. Remarkably, in wt spleens, similar amounts of
p60217–225 and LLO91–99
(ratio, 1.5) were detected, while GKO spleens yielded 30-fold less
LLO91–99 peptide than
p60217–225 peptide (ratio, 30). This difference
of GKO and wt mice occurred in the presence of similar bacterial loads
in spleen, which were 1.5 x 108 CFU and
1.2 x 108 CFU per spleen for wt and GKO
mice, respectively.
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C). The 20-fold difference of the
peptide concentration of fraction 30 from wt and GKO spleen extracts
resulted in an approximately 4-fold difference in the number of spots
per well. Remarkably, the ELISPOT assay of HPLC fractions from peptide
extracts yielded a qualitatively similar peptide presentation pattern
as the direct ELISPOT-based analysis of splenocytes isolated from
infected mice (see Fig. 1).
These results show that IFN- influences the relative abundance of
antigenic peptides in spleens, and thus changes the CD8 T cell
recognition pattern of MHC class I-restricted antigenic peptides in
L. monocytogenes-infected mice.
Effect of IFN- on the presentation of MHC class I-restricted
antigenic peptides by professional APC in vivo
Macrophages and DC are minor cell populations in the spleen, but
both play a central role in the induction of a T cell response
(20). For the direct ex vivo Ag presentation analysis of
professional APC, cells expressing CD11c or CD11b were isolated by
immunomagnetic separation from spleens of infected mice. Fig. 3 shows the cytofluorometric analysis of
cells freshly isolated by anti-CD11b- and anti-CD11c-coated
magnetic microbeads, respectively. The CD11b+
cells were always CD11c-, indicating that no
contamination with DC occurred. These anti-CD11b-selected, highly
CD11b+ cells are further referred to as
macrophages. A fraction of CD11c+ cells showed
also low level CD11b expression, which is typical for myeloid DC
(21). These highly CD11c+ cells are
further referred to as DC. The Ag presentation pattern of these
professional APC was tested qualitatively in the ELISPOT-based Ag
presentation assay (Fig. 4). To eliminate
variations due to changes of the sensitivity of T cell lines, the
different APC types were always compared in the same experiment with
the same set of T cell lines. The different peptide presentation
patterns shown were confirmed in five independent experiments using
different sets of CD8 T cell lines.
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). The comparison
of APC isolated from wt and GKO mice revealed different Ag presentation
patterns. The strongest CD8 T cell epitope presented on DC and
macrophages from L. monocytogenes-infected wt spleens was
always LLO91–99, while
p60217–225 was the strongest peptide presented
on APC isolated from GKO mice.
These data demonstrate that the presentation pattern of CD8 T cell
epitopes on professional APC is regulated by IFN-.
Analysis of the processing of MHC class I-restricted antigenic peptides in vitro
The virulence and replication of L. monocytogenes
differ strongly in wt and GKO mice. Because of the different growth
kinetics of L. monocytogenes in wt and GKO mice, it was
necessary to measure the presentation of one peptide in relation to
other peptides. This approach detected that the relative abundance of
p60217–225 and LLO91–99
in infected spleens and also the presentation of these epitopes on
professional APC are IFN- dependent. However, from these
experiments, it could not be determined whether the absence of IFN-
results in reduced generation of LLO91–99,
improved processing of p60217–225, or even both.
The quantitative measurement of the absolute effect of IFN- on the
generation of naturally processed antigenic peptides is only possible
if intracellular replication of bacteria is strictly controlled. This
could only be achieved in an in vitro infection model.
Macrophage-like P388 cells were pretreated for 24 h with 100 U
IFN- and were subsequently infected with L.
monocytogenes, and naturally processed antigenic peptides were
extracted 6 h later. Plating of infected cells on blood agar
revealed that IFN- pretreatment did not significantly inhibit
replication of bacteria during the 6-h infection phase (data not
shown). The quantification of naturally processed
p60217–225, p60449–457,
and LLO91–99 peptides revealed that IFN- had
differential effects on peptide generation (Fig. 5A). The effect of IFN- on
the generation of different peptides was calculated as the ratio of
peptides extracted in the presence and in the absence of IFN- (Fig. 5B). IFN- exerted only a minor effect on the generation
of p60217–225, but it significantly inhibited
the generation of p60449–457, and strongly
promoted the processing of LLO91–99. Similar
results were also obtained with J774 cells, another macrophage-like
cell line (data not shown).
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exerted differential, peptide-specific
effects on peptide generation in vitro that correlated well with the
peptide presentation patterns observed in wt and GKO mice.
Effect of IFN- on the presentation of MHC class II-restricted
antigenic peptides in vivo
To analyze the relevance of IFN- for the presentation of MHC
class II-restricted antigenic peptides in vivo, the peptide
presentation pattern of a number of MHC class II-restricted T cell
epitopes of L. monocytogenes was analyzed. Macrophages and
DC were isolated from spleens of mice infected for 48 h. As the
known immunodominant CD4 T cell epitopes of L. monocytogenes
are Ab restricted (22), this study
was performed with SV129 wt and IFN- receptor-deficient RKO mice
that possess the H-2b MHC haplotype. The
presentation of the MHC class II Ab-restricted
epitopes LLO190–201,
LLO318–329, LLO253–264,
and p60177–188 was analyzed qualitatively with
the ELISPOT-based ex vivo Ag presentation assay using peptide-specific
CD4 T lines to detect the presentation of specific peptides by
professional APC (Fig. 6). Macrophages
and DC isolated 48 h after infection with 1 x
104 CFU revealed similar Ag presentation patterns
of CD4 T cell epitopes. The strongest peptide presented on both cell
types was LLO190–201. The second strongest
peptide presented on DC was p60177–188. The
second strongest peptide presented on wt macrophages was
LLO253–264. The peptide presentation pattern of
DC from wt mice (LLO190–201 >
p60177–199 > LLO318–329
= LLO253–264) was similar on DC isolated from
RKO mice (Fig. 6, left). Compared with the Ag presentation
pattern of macrophages from wt mice (LLO190–201
> LLO 253–264 >
p60177–199 = LLO318–329),
macrophages isolated from RKO mice revealed weaker presentation of LLO
253–264 (Fig. 6, right). However, presentation of LLO
253–264, p60177–199, and
LLO318–329 was generally weak on macrophages,
and the observed small differences between these peptides were not
constant. The general Ag presentation pattern of CD4 T cell epitopes
was observed in three independent experiments. Generally, the overall
strength of Ag presentation was reduced on DC and macrophages isolated
from infected RKO mice. In summary, these results demonstrate that
despite the fact that presentation of L.
monocytogenes-derived CD4 T cell epitopes by professional APC in
vivo was enhanced by IFN-, it was not generally required for
efficient MHC class II-restricted Ag presentation, and also did not
change the presentation pattern of different CD4 T cell epitopes.
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Remarkably, strong presentation of CD4 T cell epitopes on
macrophages and DC occurred in the absence of IFN-. To investigate
the effect of IFN- on the general MHC expression level, a
cytofluorometric analysis of MHC class I and MHC class II expression
was performed. DC were isolated either from naive SV 129 wt and RKO
mice or 48 h after L. monocytogenes infection (Fig. 7). Generally, MHC class I and II
expression was stronger on APC from wt compared with RKO mice. However,
remarkably, a significant up-regulation of MHC class II expression
occurred also after infection of RKO mice, indicating that
up-regulation of MHC expression is at least in part IFN-
independent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The stability of MHC class I/peptide complexes strongly influences the abundance of naturally processed antigenic peptides in L. monocytogenes-infected cells if the supply of Ag is limited (28). LLO91–99 and p60217–225 form relatively stable peptide/Kd complexes with a t1/2 of approximately 6 h, while p60449–457/Kd complexes have a t1/2 of less than 1 h (28, 29). Thus, the peptide presentation pattern of CD8 T cell epitopes, i.e., strong presentation of LLO91–99 and p60217–225 and weak presentation of p60449–457, reflects the stability of peptide/MHC class I complexes. The same Ag presentation pattern was also obtained with macrophages and unseparated spleen cells in the current and also a previous study (15), suggesting that presentation of L. monocytogenes-derived CD8 T cell epitopes by all important APC populations occurs under conditions in which the supply of Ag is limiting.
The analysis of L. monocytogenes-infected spleens showed
that IFN- had only a peptide-specific influence on the presentation
pattern of CD8 T cell epitopes in vivo, and that the lack of IFN-
was not generally accompanied with reduced MHC class I-restricted Ag
presentation. Previously, it was shown that IFN- has a general
enhancing effect on MHC class I- and MHC class II-restricted Ag
presentation by L. monocytogenes-infected macrophages in
vitro (30). A general enhancing effect of IFN- on Ag
presentation has been shown also in an in vivo study of mice infected
with the murine CMV (31).
IFN- modulates the presentation of CD8 T cell epitopes by a number
of different mechanisms. It modulates the cleavage preferences of the
proteasome, influences the TAP-mediated transport of antigenic
peptides, and also up-regulates the expression of MHC class I molecules
(9, 10, 11). The analysis of the effect of IFN- on the
processing and presentation of different L.
monocytogenes-derived peptides in vivo revealed that it improved
processing of one peptide (LLO91–99), reduced
processing of another (e.g., p60449–457), or had
no effect at all (p60217–225). These
differential, peptide-specific effects of IFN- on Ag processing in
vitro correlate with the effect of IFN- in vivo. The changed
cleavage specificity of the IFN- -inducible immunoproteasome
provides a possible explanation for these IFN--mediated,
peptide-specific effects on the Ag presentation pattern
(32, 33, 34, 35, 36). Dependent on the sequence of the epitope itself
and also its flanking sequences, the changed cleavage specificity of
the immunoproteasome could mediate differential effects on the
processing of individual antigenic peptides.
Professional, bone marrow-dependent APC are required for the induction
of a primary antilisterial CD8 T cell response (37). Thus,
the IFN--mediated regulation of the peptide presentation pattern of
DC and macrophages could influence the hierarchy of responding CD8 T
cell populations. From studies of the murine L.
monocytogenes (3, 8) and the lymphocytic
choriomeningitis virus infection (38), it is known that
IFN- is not required to induce a specific CD8 T cell response and
that the infection of GKO mice even results in an enhanced
peptide-specific CD8 T cell response. This apparently paradoxical
situation could be due to a higher bacterial or viral load in GKO mice,
as it is clearly the situation in lymphocytic choriomeningitis
virus-infected GKO mice (38). The analysis of the
hierarchy of T cell populations in GKO and wt mice showed a changed
immunodominance pattern of LLO91–99- and
p60217–225-specific CD8 T cell populations
(8). While in wt mice
LLO91–99-specific CD8 T cells outnumbered
p60217–225-specific cells in a 5:1 ratio, in GKO
mice p60217–225- and
LLO91–99-specific CD8 T cells were present in a
2:1 ratio. Remarkably, this change of the immunodominance pattern of
antilisterial CD8 T cell populations correlates with the observed
effect of IFN- on the peptide presentation pattern of ex vivo
isolated DC and macrophages.
In contrast to the CD8 T cell epitopes, no strong influence of IFN-
on the peptide presentation pattern of CD4 T cell epitopes was
observed. However, presentation of CD4 T cell epitopes was generally
weaker in RKO mice. This effect correlated with a generally reduced MHC
class II expression level on APC from RKO mice. Remarkably, the
up-regulation of MHC class II molecules after infection occurred also
in RKO mice, suggesting that IFN--independent up-regulation of MHC
molecules might act compensatory in RKO mice.
The stability of MHC class II/peptide complexes of L. monocytogenes-derived CD4 T cell epitopes is currently unknown. However, similar to the CD8 T cell epitopes, the presentation of CD4 T cell epitopes also showed a typical peptide presentation pattern. LLO190–201 was always the strongest peptide detected on DC and macrophages. This epitope is also the immunodominant CD4 T cell epitope in L. monocytogenes-infected C57BL/6 mice (22). Thus, a similar correlation exists as for CD8 T cell epitopes, in which the immunodominant CD8 T cell populations are directed against the most stable epitopes that are most abundant in vivo (15).
In summary, these results show that despite IFN- modulation of the
Ag presentation pattern of CD8 T cell epitopes, IFN- is not
generally required for the MHC class I- and MHC class II-restricted
presentation of L. monocytogenes-derived antigenic peptides
by professional APC in vivo. Considering the multiple steps of Ag
processing and presentation influenced by IFN-, this is an
unexpected result. The constitutive ability of macrophages and DC to
efficiently process and present Ags highlights the functional
properties of these dedicated APC. The full appreciation of these
functional properties requires further detailed analysis on how
professional APC acquire and process Ags in vivo.
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Acknowledgments |
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Footnotes |
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. was supported by the Slovenian Ministry of Science and the Medical Faculty of the University of Ljubljana. 
2 Address correspondence and reprint requests to Dr. Gernot Geginat, Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail address: geginat{at}rumms.uni-mannheim.de
3 Abbreviations used in this paper: GKO, IFN- knockout; DC, dendritic cell; LLO, listeriolysin O; p.i., postinfection; RKO, IFN- receptor knockout; wt, wild type.
Received for publication October 11, 2001. Accepted for publication December 7, 2001.
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References |
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  K. A. N. Messingham, V. P. Badovinac, A. Jabbari, and J. T. Harty A Role for IFN-{gamma} from Antigen-Specific CD8+ T Cells in Protective Immunity to Listeria monocytogenes J. Immunol., August 15, 2007; 179(4): 2457 - 2466. [Abstract] [Full Text] [PDF]
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T. R. Kollmann, B. Reikie, D. Blimkie, S. S. Way, A. M. Hajjar, K. Arispe, A. Shaulov, and C. B. Wilson Induction of Protective Immunity to Listeria monocytogenes in Neonates J. Immunol., March 15, 2007; 178(6): 3695 - 3701. [Abstract] [Full Text] [PDF]
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M. Skoberne, S. Schenk, H. Hof, and G. Geginat Cross-Presentation of Listeria monocytogenes-Derived CD4 T Cell Epitopes J. Immunol., August 1, 2002; 169(3): 1410 - 1418. [Abstract] [Full Text] [PDF]
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) and DC is shown. Cells were stained with
Abs of the indicated specificity (bold lines) or with isotype control
Abs (thin lines).
) or left untreated (
). After HPLC separation, fractions
containing antigenic activity were identified in a 51Cr
release assay with p60217–225-, p60449–457-,
and LLO91–99-specific CD8 T cells, respectively. The total
peptide content of positive fractions was determined by linear
interpolation from a synthetic peptide standard. Shown is the total
number of naturally processed antigenic peptides per L.
monocytogenes-infected cell from a representative experiment.
B, The ratio of peptides (p60217–225,
p60449–457, and LLO91–99) extracted from
IFN-
