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Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production and
Listeria-specific cytotoxicity. During primary
Listeria infection of CD28-/- mice we
observed significantly reduced numbers of
Listeria-specific CD8+ T cells and only
marginal levels of specific IFN- production and cytotoxicity.
Although frequencies were also reduced in CD28-/- mice
during secondary response, we detected a considerable population of
Listeria-specific CD8+ T cells in these
mice. In parallel, IFN- production and cytotoxicity were observed,
revealing that Listeria-specific CD8+ T
cells in CD28-/- mice expressed normal effector
functions. Consistent with their impaired CD8+ T cell
activation, CD28-/- mice suffered from exacerbated
listeriosis both after primary and secondary infection. These results
demonstrate participation of CD28 signaling in the generation and
expansion of Ag-specific CD8+ T cells in listeriosis.
However, Ag-specific CD8+ T cells generated in the absence
of CD28 differentiated into normal effector and memory T
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1.5% of the CD8+ T cells were
specific for the listeriolysin O (LLO)4-derived peptide
LLO91–99, the most dominant epitope
analyzed. During the secondary response,
LLO91–99-specific T cells reached levels higher
than 15% of all CD8+ T cells (5).
Together with our results from transfer experiments (3),
these findings demonstrate that an unexpectedly large fraction of
CD8+ T cells is involved in the antilisterial
response. Ag recognition by the TCR induces activation of T lymphocytes. However, TCR-mediated signals alone are insufficient for efficient T cell activation and additional costimulatory signals are required. One of the most important surface molecules that delivers costimulatory signals for T cells is CD28. CD28 is expressed on T cells, NK cells, and ligands for CD28 and the structurally related CTLA4 (CD152) are the molecules B7.1 (CD80) and B7.2 (CD86) (6). B7.1 and B7.2 molecules are expressed on professional APCs and their expression is up-regulated during an immune response (6). Stimulation of CD4+ T cells in the absence of CD28-mediated cosignaling causes impaired proliferation, reduced cytokine production, and altered generation of helper T cell subsets (7, 8, 9). The role of CD28 costimulation in the activation and differentiation of CD8+ T cells is less clear and the results of different studies are in part contradictory. In diverse in vitro models, T cells were stimulated under controlled costimulatory conditions either with defined APC or, in an APC-independent fashion, with anti-TCR mAb or purified MHC class I molecules. Results of these studies vary from strict dependence (10, 11, 12, 13) partial requirement (14, 15, 16) to complete independence of CD28 costimulation for the activation and differentiation of CD8+ T cells (17, 18, 19, 20). A possible explanation for at least part of these contradictory results is given by the observation that CD28 costimulation reduces the number of TCRs that have to be triggered to achieve T cell activation (21, 22). Consistent with this observation, the need for costimulatory signals was particularly manifested under conditions with limited TCR signals (14, 15, 17, 18, 20, 23). Furthermore, requirement on CD28 costimulation was influenced by the differentiation state of CD8+ T cells, because preactivated CD8+ T cells were less dependent on CD28 (13, 18).
The contribution of CD28 to the activation of CD8+ T cells in vivo is even more controversial. Treatment of mice with nonimmunogenic tumors that had been transfected with one of the B7 molecules resulted in antitumor responses and protection against subsequent challenge with untransfected tumors (24, 25, 26, 27, 28). Blocking of CD28 signaling with a soluble CTLA4 protein prolonged graft survival and induced donor specific tolerance (29, 30, 31). However, more recent experiments using different B7-transfected tumors failed to demonstrate a protective antitumor response (32, 33, 34, 35). Furthermore, CD28-/- mice respond to syngeneic or induced tumors (36) and reject allografts (37). In different viral infection models the requirement for CD28 signaling to generate a virus-specific CD8+ T cell response also varied significantly. Whereas the CD8+ T cell response of mice against infection with lymphocytic choriomeningitis virus (LCMV) was completely independent of CD28 signaling (7, 38, 39), CD8+ T cell responses against vesicular stomatitis virus (VSV), influenza virus, and certain strains of vaccinia virus were drastically impaired (7, 38, 39, 40, 41, 42). For viral infections, several mechanisms have been suggested to explain the distinct CD28 requirements, including differences in viral load, viral persistence, and the degree of inflammation (43, 44).
We infected CD28-deficient mice with L. monocytogenes and
analyzed their immune responses, with the main emphasis on the
generation and differentiation of Listeria-specific
CD8+ T cells as the major mediators of acquired
resistance. We show that CD28 is important for the protective
antilisterial response, as demonstrated by higher bacterial titers in
the liver and the spleen, delayed clearance of bacteria, and increased
susceptibility to infection in CD28-/- mice.
During primary infection, Listeria-specific cytotoxicity and
frequencies of IFN- secreting Listeria-specific
CD8+ T cells were markedly diminished. A closer
analysis with MHC class I tetramers loaded with dominant listerial
peptides revealed significantly reduced numbers of
Listeria-specific CD8+ T cells. During
the secondary response we identified a significant population of
Listeria-specific CD8+ T cells and, in
close correlation, a substantial population of
CD8+ effector T cells. Thus, the lack of CD28
impairs the generation, and particularly the expansion, of specific
CD8+ T cells during L. monocytogenes
infection; however, it does not prevent differentiation into
CD8+ effector and memory T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28-/- mice, backcrossed eight times onto the BALB/c background, were kindly provided by Dr. T. W. Mak (Toronto, Canada) (7). CD28-/- mice and CD28+/+ BALB/c control mice were bred in our facility at the Federal Institute for Health Protection of Consumers and Veterinary Medicine (Berlin, Germany) and experiments were conducted according to the German animal protection law.
Bacteria and bacterial infection of mice
Mice were infected with L. monocytogenes strain EGD. Bacteria were grown overnight in tryptic soy broth (TSB), washed twice in PBS, aliquoted in PBS 10% glycerol, frozen, and stored at -80°C. Aliquots were thawed and bacterial titers were determined by plating serial dilutions on TSB agar plates. For infection, aliquots were thawed and appropriately diluted in PBS. Bacteria were injected in a volume of 200 µl PBS into the lateral tail veins of mice. The bacterial dose was controlled by plating dilutions of the inoculum on TSB agar plates. For determination of bacterial burdens in organs, mice were killed at the time points indicated. Livers and spleens were homogenized in PBS, serial dilutions of homogenates were plated on TSB agar plates, and colonies were counted after incubation at 37°C overnight. Unless otherwise indicated, mice were first infected with 103 bacteria. After 2–3 mo, mice were secondarily infected with 105 bacteria. In our hands, LD50-values for primary i.v. infection with L. monocytogenes were 8.5 x 103 and 4.0 x 103 Listeria for CD28+/+ and CD28-/- mice, respectively.
Antibodies
Polyclonal rat Abs, anti-CD16/CD32 mAb (clone, 2.4G2),
anti-IFN- mAb (clone, R4-6A2, rat IgG1), anti-CD3 mAb
(clone, 145 2C11), anti-CD4 mAb (clone, YTS191.1), anti-CD8
mAb (clone, YTS169), and anti-CD62L mAb (clone, Mel-14) were
purified from rat serum or hybridoma supernatants with protein
G-Sepharose. Abs were Cy5- or FITC-conjugated according to standard
protocols. PE-conjugated anti-IL-2 mAb (clone, JES6-5h4, rat
IgG2b), FITC-conjugated rat IgG1 isotype control mAb (clone, R3-34),
and PE conjugated rat IgG2b isotype control mAb (clone, A95-1) were
purchased from BD PharMingen (San Diego, CA).
In vitro restimulation of spleen cells and flow cytometric
determination of IFN- expression
At the time points indicated, spleens from infected mice were removed and single cell suspensions were obtained by teasing spleens through stainless steel meshes. Erythrocytes were lysed and spleen cells (3 x 106/well) were cultured in 48-well plates in RPMI medium supplemented with glutamine, Na-pyruvat, 2-ME, penicillin, streptomycin, and 10% heat inactivated FCS. Spleen cells were stimulated for 6 h with 5 µg/ml anti-CD3 mAb or with 10-6 M of the peptides LL091–99 (GYKDGNEYI) or p60217–225 (KYGVSVQDI), both purchased from Jerini Bio Tools (Berlin, Germany). During the final 4 h of culture, 10 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) were added.
Cultured cells were washed and incubated with polyclonal rat Abs and
anti-CD16/CD32 mAb to block unspecific Ab binding. After 10 min,
cells were stained with Cy5-conjugated anti-CD4 mAb or
anti-CD8 mAb. After 30 min on ice, cells were washed with PBS
and fixed for 20 min at room temperature with PBS 4% paraformaldehyde
(Sigma-Aldrich). Cells were washed with PBS, 0.1% BSA, permeabilized
with PBS 0.1% BSA, 0.5% saponin (Sigma-Aldrich) and incubated in this
buffer with polyclonal rat Abs and anti-CD16/CD32 mAb. After 5 min,
FITC-conjugated anti-IFN- mAb or FITC-conjugated isotype control
mAb was added. After an additional 20 min at room temperature, cells
were washed with PBS and fixed with PBS 1% paraformaldehyde. Cells
were analyzed using a FACSCalibur and the CellQuest software (BD
Biosciences, Mountain View, CA).
Generation of MHC class I tetramers and staining of cells with tetramers
Modified forms of the full-length cDNA of
H-2Kd and human 
2m were
kindly provided by Drs. E. Pamer and D. Busch. Tetrameric
H-2Kd/peptide complexes were generated as
described by Busch et al. (5). Briefly, human
2m and the extracellular domains of
H-2Kd fused with a peptide containing a specific
biotinylation site were expressed as recombinant proteins in E.
coli. Proteins were purified, dissolved in 8 M urea, and diluted
into a refolding buffer containing the peptides
LLO91–99 or p60217–225 to
generate monomeric soluble MHC class I-peptide complexes. These
complexes were purified by gel filtration and enzymatically
biotinylated using the biotin protein ligase BirA (Avidity,
Denver, CO). Free biotin was removed and MHC-peptide complexes were
purified by gel filtration. To generate tetrameric MHC-peptide
complexes, PE-conjugated streptavidin (Molecular Probes, Eugene, OR)
was added to the monomers at a molar ratio of 4:1. Tetramers were
purified by gel filtration and stored at 4°C.
For flow cytometry analysis, 1 x 106 cells
were incubated for 15 min at 4°C with polyclonal rat Abs,
anti-CD16/CD32 mAb, and streptavidin (Molecular Probes) in PBS
containing 0.5% BSA and 0.01% sodium azide. After incubation, cells
were stained for 60 min at 4°C with Cy5-conjugated anti-CD8
mAb, FITC-conjugated anti-CD62L mAb, and PE-conjugated MHC class
I-LLO91–99 or MHC class
I-p60217–225 tetramers. Subsequently, cells were
washed with PBS 0.5% BSA, 0.01% sodium azide, and diluted in PBS.
Propidium iodide was added before 4-color flow cytometry analysis.
IFN- production after restimulation in vitro
Spleens from infected mice were removed and 5 x
105 cells/well were cultured in 96-well plates in
triplicate per experimental value and mouse. Cells were stimulated with
5 µg/ml anti-CD3 mAb or with 10-6 M of the
peptides LLO91–99 or
p60217–225. After 2 days, IFN- was determined
in the supernatants by ELISA as described previously
(45).
Cytotoxicity assay
At the time points indicated, spleens from infected mice were removed and 3 x 106 cells were cultured in 10 ml complete medium supplemented with 30 U of recombinant human IL-2. Cells were restimulated with 3 x 106 naive BALB/c splenocytes that had been either loaded with 10-7 M of the peptides LLO91–99 or p60217–225 or were left untreated. After 3 days, cells were washed, counted, and incubated with 5000 51Cr-labeled P815 target cells at the effector/target ratios indicated in V-bottom plates in a total volume of 200 µl. Peptides were added at 10-6 M. After 4 h, supernatants (100 µl) were counted with a gamma counter. Each value was determined as triplicate. Cytotoxicity is given as the percentage of specific lysis which was calculated with the standard formula: percentage of specific lysis = 100 x (experimental 51Cr release - spontaneous 51Cr release)/(detergent induced 51Cr release - spontaneous 51Cr release).
Statistical analysis
The statistical significance of the results was determined with the statistics program included in the GraphPad Prism program (GraphPad, San Diego, CA). Mean bacterial titers are given as the geometric mean and differences in titers were determined with the unpaired t test from log-transformed values: *, p < 0.05; **, p < 0.005.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28-/- and control mice were i.v.
infected with 2 x 103 L.
monocytogenes and bacterial titers in spleens and livers were
determined (Fig. 1
). Control mice had
high bacterial titers on day 3 of infection but then readily cleared
bacteria. In contrast, CD28-/- mice had 10- to
100-fold higher bacterial titers in spleens and livers on days 3 and 6
of infection. By day 9, most CD28+/+ mice had
cleared the infection, whereas CD28-/- mice
still suffered from considerable bacterial loads. L.
monocytogenes infection did not result in chronic disease in
CD28-/- mice and on day 13 most
CD28-/- mice were free of bacteria. Increased
listerial titers caused death in a significant proportion of
CD28-/- mice. Although, we lowered the dose to
103 Listeria in all additional
experiments, still 20–25% of the CD28-/- mice
succumbed to infection whereas none of the control mice died from
this dose.
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). Two days and particularly 3 days
after challenge with 105 L.
monocytogenes we observed elevated bacterial titers in spleens and
livers of CD28-/- mice compared with control
mice. However, there was significant protection in challenged
CD28-/- mice when compared with naive
CD28-/- mice, indicating that CD28 is not
absolutely required for protective immunity against L.
monocytogenes.
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T cells are central for acquired immunity against infection with L. monocytogenes. Particularly CD8+ T cells are critical for control and eradication of this pathogen (2). For BALB/c mice several immunodominant CD8+ T cell epitopes from L. monocytogenes have been characterized (5), which allowed us to analyze the generation of the Ag-specific CD8+ T cell response against L. monocytogenes in detail. For our additional experiments, we used the peptides LLO91–99 and p60217–225 which are both presented by H-2Kd.
In a first series of experiments, we investigated whether
CD28-/- mice were able to mount a
Listeria-specific CTL response. Spleen cells from infected
mice were restimulated with LLO91–99 or
p60217–225 for 3 days. Cultures contained IL-2
to supplement the reduced cytokine production of CD28-deficient T cells
(7). Peptide-specific cytotoxicity was tested in a 4
h 51Cr release assay (Fig. 3). In CD28+/+
mice, infection with L. monocytogenes induced a CTL response
against both listerial peptides during the primary response and
cytotoxicity was enhanced during the secondary response. In
CD28-/- mice, only very low levels of
peptide-specific cytotoxicity were measured during the primary
response. During the secondary response, specific cytotoxicity was
detected although at a reduced level compared with
CD28+/+ mice.
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Our cytotoxic assay depends on in vitro restimulation of specific
CD8+ T cells. Although we cultured these cells
for only 3 days and added IL-2, we cannot formally exclude that
differences in the CTL assays were due to altered behavior of
CD28-deficient T cells in culture. Furthermore, the CTL assay does only
allow limited frequency estimates of responding
CD8+ T cells. Therefore, we generated MHC class
I-peptide tetramers containing the peptides
LLO91–99 or p60217–225 to
quantify Listeria-specific CD8+ T
cells directly ex vivo. With both tetramers we detected activated
CD8+CD62Llow T cells in
CD28+/+ control mice infected with L.
monocytogenes (Fig. 4).
Peptide-specific T cells were first identified at days 4 and 5 and the
response reached a maximum at days 9 and 10. At the peak of response,
spleens from infected mice contained 3 x
105 LLO91–99 specific
CD8+CD62Llow T cells and
1 x 105
p60217–225 specific
CD8+CD62Llow T cells (Fig. 5). Although Listeria-specific
CD8+ T cells were generated in
CD28-/- mice, their numbers were markedly
reduced, particularly at the peak of response on days 9 and 10. Only at
later time points of infection were levels of specific
CD8+CD62Llow T cells
comparable in CD28+/+ and
CD28-/- mice. These results imply that the lack
of CD28 does not principally block induction of
Listeria-specific CD8+ T cells.
However, generation, and particularly expansion, of these cells during
the peak of response is significantly impaired.
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In CD28+/+ mice, secondary Listeria
infection caused a marked expansion of the Listeria-specific
CD8+ T cell population. At the peak of secondary
response, 20% of the CD8+ T cells were
LLO91–99 tetramer+ and
4–5% were p60217–225
tetramer+, correlating with 2 x
106 and 4 x 105 of
LLO91–99 and p60217–225
specific CD8+ T cells per spleen, respectively
(data not shown and Fig. 6). In
CD28-/- mice, the secondary response of
specific CD8+ T cells showed a kinetic equivalent
to that observed in CD28+/+ mice. Similar to the
primary response, the magnitude of the secondary response was reduced
in CD28-/- mice compared to
CD28+/+ mice. However,
CD28-/- mice developed a memory response
because numbers of Listeria-specific
CD8+ T cells were considerably increased compared
to numbers in the primary response. Thus, frequencies of Ag-specific
CD8+ T cells were diminished in the absence of
CD28 in both the primary and secondary responses to L.
monocytogenes.
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secreting CD8+ T cells
in L. monocytogenes-infected CD28-/- mice
In addition to MHC class I tetramer staining, we measured IFN-
production after short term in vitro restimulation with peptides to
determine frequencies of Listeria-specific
CD8+ T cells. In contrast to tetramer staining,
this assay is based on cytokine production and therefore allows an
estimate of the frequencies of specific CD8+
effector T cells. CD28+/+ and
CD28-/- mice were infected with L.
monocytogenes. On days 7 and 10 of primary infection and on days 5
and 7 of secondary infection (Fig. 7),
frequencies of IFN- producing CD8+ T cells
were determined by intracellular cytokine staining after short-term
restimulation with LLO91–99. During both primary
and secondary responses, we observed Ag-specific
CD8+ T cells in both
CD28+/+ and CD28-/- mice.
However, the frequencies of IFN- producing T cells were markedly
reduced in CD28-/- mice, particularly during
the primary response. Restimulation with
p60217–225 resulted in 4- to 5-fold lower
frequencies compared to restimulation with
LLO91–99 but a similar difference of response
between CD28+/+ and
CD28-/- mice (data not shown). When compared at
a single cell level, IFN-+
CD8+ T cells from CD28+/+
and CD28-/- mice showed the same intensity of
anti-IFN- mAb staining, indicating that single IFN--positive
CD28+/+ and CD28-/-
CD8+ T cells produced similar amounts of IFN-
(Fig. 8 and data not shown).
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production was also observed by ELISA.
Spleen cells of mice infected for 8 days with L.
monocytogenes were restimulated in vitro for 2 days with peptides
and IFN- in the supernatants was determined. Cells from infected
CD28+/+ mice produced 14.8 ± 2.6 and
7.3 ± 5.3 U IFN-/ml after restimulation with
LLO91–99 or p60217–225,
respectively. IFN- production of cells from naive mice and infected
CD28-/- mice was below the detection limit of 1
U/ml (mean ± SD from 3 mice per group; three independent
experiments).
IFN- and IL-2 secretion of CD4+ and CD8+
T cells from CD28+/+ and CD28-/- mice
infected with L. monocytogenes
It could be argued that the reduced frequencies of
Listeria-specific CD8+ T cells was due
to limited production of growth factors resulting in impaired expansion
of the specific CD8+ T cell population. IL-2
production was analyzed in CD8+ T cells from
L. monocytogenes infected mice after short-term peptide
restimulation (Fig. 8
). Although a high frequency of
CD8+ T cells from CD28+/+
mice secreted IFN- after peptide restimulation, there was hardly any
IL-2 secretion by this cell population in our 6 h assay. As
described above, frequencies of IFN- secreting
CD8+ T cells were significantly reduced in
spleens from CD28-/- mice. Polyclonal
restimulation with soluble anti-CD3 mAb lead to similar results. We
observed IFN- secreting CD8+ T cells in
infected CD28+/+ and
CD28-/- mice, but there were only very low
frequencies of IL-2 secreting CD8+ T cells in
spleens of these mice (data not shown).
IL-2 and IFN- secretion by CD4+ T cells was
also analyzed. Because strong immunodominant CD4+
T cell epitopes from L. monocytogenes have not been
characterized for BALB/c mice, we used polyclonal anti-CD3 mAb
restimulation for induction of cytokine secretion in
CD4+ T cells (Fig. 8). In spleen cells from naive
CD28+/+ mice, anti-CD3 stimulation induced a
high frequency of cells secreting IFN- and/or IL-2. After infection
of mice, cytokine-positive populations were enlarged and this effect
was most prominent for the IFN-/IL-2 double-positive population. In
contrast, there was reduced IL-2 and IFN- production in spleen cells
from CD28-/- mice and frequencies did not
increase after infection of these mice with L.
monocytogenes. Reduced IFN- production in infected
CD28-/- mice was also evident when spleen cells
from naive and infected CD28+/+ and
CD28-/- mice were restimulated for 2 days with
anti-CD3 mAb and IFN- production was determined by ELISA (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. However, Ab treated and control mice showed equal
listerial titers in livers and spleens during primary and secondary
infection. We observed markedly diminished IFN- production after in
vitro restimulation of spleen cells from infected
CD28-/- mice, but lack of CD28 signaling
impaired resistance to L. monocytogenes infection in our
experiments. There are several explanations for these conflicting
results. Compared with a genetic CD28-deficiency, injection of
anti-B7 mAb could lead to only partial inhibition of CD28 signaling
and anti-B7 mAb could affect not only CD28 but also CTLA4
signaling. Furthermore, differences in virulence of L.
monocytogenes strains and susceptibility of mouse strains (C57BL/6
vs BALB/c) cannot be excluded. In CD28+/+ mice, analysis of LLO91–99 and p60217–225-specific CD8+ T cells with MHC class I tetramers gave results similar to those described by Busch et al. (5), although in our experiments, the response was slightly delayed which can be explained by differences in the virulence of the L. monocytogenes strains used. In CD28-/- mice, we detected Ag-specific CD8+ T cells but at significantly reduced levels, particularly during the primary response. Furthermore, the profile of the primary response was altered in that CD8+ T cell frequencies did not decline at late time points of infection as was the case in control mice. The delayed clearance of L. monocytogenes in CD28-/- mice should result in increased amounts of Ag available for prolonged time periods, which could in turn induce continued T cell stimulation. In addition, the sustained bacterial persistence could maintain an inflammatory environment that could support prolonged survival and expansion of Listeria-specific CD8+ T cells in CD28-/- mice.
How could the lack of CD28 signaling affect the generation and
expansion of Ag-specific CD8+ T cells? Several
reports indicate that the deficiency of CD28 costimulation does not
impair early events of T cell activation in vitro and in vivo, but
without costimulation T cell activation is incomplete and cannot be
sustained resulting in reduced proliferation, induction of
unresponsiveness, and increased levels of apoptosis. These defective
responses could be related to reduced production of growth factors and
impaired induction of antiapoptotic molecules such as
BCL-xL (7, 8, 44, 48, 49, 50, 51). So far,
we have no evidence for increased T cell apoptosis in our infection
model and the prolonged listerial persistence and consequently
sustained T cell stimulation does not allow valid comparisons on T cell
survival in our model. We regard reduced growth factor production as a
major factor responsible for the impaired generation, and especially
expansion, of CD8+ T cells. In this context, it
is important to consider that the lack of CD28 costimulation affects
not only CD8+ T cells but also
CD4+ T cells and other cell types that normally
express CD28. Flow cytometric analysis of T cells that had been
restimulated with anti-CD3 mAb revealed reduced frequencies of
IFN- and IL-2 secreting CD4+ T cells in
infected CD28-/- mice, indicating that
generation and expansion of Listeria-specific
CD4+ T cells is impaired, as well. In our assays,
CD8+ T cells produce only marginal amounts of
IL-2. In contrast, CD4+ T cells are a major
source of this growth factor and reduced IL-2 production by
CD4+ T cells could at least in part explain the
impaired expansion of the Listeria-specific
CD8+ T cell population. It has been shown that
CD4+ T cells are involved in protection against
L. monocytogenes. However, their role in the generation of
Listeria-specific CD8+ T cells needs
further investigation.
Characterization of the CD8+ T cell response in CD28-/- mice during secondary infection demonstrated that in terms of kinetics and magnitude, this response showed all the hallmarks of a secondary T cell response. Similar to the situation in CD28+/+ mice, the kinetic of the secondary response in CD28-/- mice was accelerated and, at the peak of the secondary response, the numbers of peptide-specific CD8+ T cells were 5- to 10-fold higher when compared to the numbers at the peak of the primary response. In addition, we were able to directly identify Listeria-specific CD8+ T cells in both CD28+/+ and CD28-/- mice 2–3 mo postinfection. In summary, we demonstrate that CD28-/- mice can generate CD8+ memory T cells at a level that is not drastically reduced compared with CD28+/+ mice. This result also indicates that the capacity of CD28 costimulation to prolong T cell survival is not essential for the generation and maintenance of CD8+ memory T cells. The results of our L. monocytogenes infection model are consistent with data from viral models showing that impaired CD28 signaling does not abrogate generation of virus-specific CD8+ memory T cells (38, 39, 40).
To evaluate the role of CD28 costimulation in the differentiation of
Ag-specific CD8+ T cells into effector cells, we
analyzed cytotoxicity against peptide-loaded cells and IFN-
production by single cells. We detected significant
Listeria-specific cytotoxicity in
CD28-/- spleen cells only after secondary
L. monocytogenes infection. Considering the low frequencies
of Listeria-specific CD8+ T cells
identified with the tetramer analysis, this observation is consistent
with the interpretation that CD28 signaling is not essential for the
differentiation of Listeria-specific
CD8+ T cells into CTL and only during secondary
infection do frequencies of these cells exceed the threshold of our CTL
assay. However, this result should be evaluated with care because
spleen cells were cultured for 3 days in the presence of IL-2. IL-2
could bypass the requirement for CD28 costimulation and thereby allow
the differentiation of CD8+ T cells into CTL
during in vitro culture. The analysis of IFN- production on the
single-cell level is more conclusive because we used only short-term
culture without additional exogenous cytokines. Frequencies of IFN-
secreting CD8+ T cells were in close correlation
to frequencies of T cells determined with tetramers. This result
confirms our assumption that CD28 costimulation is not a prerequisite
for the differentiation of CD8+ T cells and that
the low numbers of CD8+ effector T cells observed
in CD28-/- mice are due to the lowered
frequency of Listeria-specific T cells.
When directly compared, frequencies of tetramer+
CD8+ T cells were 2- to 3-fold higher than
frequencies of IFN-+
CD8+ T cells responding to the specific peptide
and this difference was maintained during the course of infection in
both CD28+/+ and CD28-/-
mice. One explanation is that only a fraction of the
LLO91–99 and
p60217–225-specific CD8+ T
cells secreted IFN-. It is also possible that the lower frequencies
of IFN-+ CD8+ T cells
are due to the limits of our assay. Although peptide restimulation
lasted for only 6 h, we cannot exclude the fact that a proportion
of specific T cells died as a consequence of activation-induced cell
death. Current experiments are aimed to clarify this difference in
detail.
When compared with different viral infection systems, our results from
the Listeria system are comparable to VSV, influenza virus,
and low virulence strains of vaccinia virus, but different to LCMV and
high virulence strains of vaccinia virus (7, 38, 39, 40, 41, 42). In
the former group of viral infections, blocking of CD28 costimulation
results in diminished CTL responses and reduced numbers of
virus-specific IFN--secreting CD8+ T cells. In
contrast, the response to LCMV and high virulence strains of vaccinia
virus appeared to be not impaired or only marginally impaired. It has
been suggested that differential requirements for CD28 costimulation in
these models were at least in part due to the level of Ag load and Ag
persistence during infection (43, 44). Viruses such as
LCMV and virulent strains of vaccinia virus that replicate widely in
mice produce abundant amounts of viral Ags for a sustained time period.
In contrast, VSV replicates abortively and viral Ags are only available
in small amounts for a limited time period. As a consequence, CD28
costimulation would be more important for the generation and expansion
of VSV-specific CD8+ T cells (43, 44). Our results imply that this hypothesis cannot be
generalized to all types of infections. CD28-/-
mice suffered from high listerial titers over an extended period of
time. Therefore, large amounts of Ag should be available for sustained
activation of CD8+ T cells. However, the
CD8+ T cell response was still impaired,
indicating that factors other than abundance and availability of Ag
influence the dependence on CD28 costimulation.
As an alternative explanation it has been proposed that pattern recognition by the innate immune system creates an environment that supports activation and expansion of Ag-specific T cells in the absence of costimulatory molecules (43). L. monocytogenes infection causes a massive inflammation; listerial cell wall components such as lipoteichoic acids have been shown to activate macrophages via Toll-like receptors and to induce the production of proinflammatory cytokines (52). Together with additional costimulatory molecules such as inducible costimulatory molecule, heat stable Ag, or members of the TNFR family (reviewed in Ref. 53), inflammatory cytokines and other factors of the innate immune system are certainly involved in the residual generation, expansion, and differentiation into effector or memory CD8+ T cells that occurs in CD28-/- mice. Yet, the impaired CD8+ T cell response to L. monocytogenes implies that CD28 fulfils unique functions that cannot be compensated for by other mechanisms of the innate or acquired immune system.
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Acknowledgments |
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Footnotes |
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2 H.-W.M. and M.K. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Hans-Willi Mittrücker, Department of Immunology, Max Planck Institute for Infection Biology, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail address: mittruecker{at}mpiib-berlin.mpg.de
4 Abbreviations used in this paper: LLO, listeriolysin O; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular stomatitis virus; TSB, tryptic soy broth.
Received for publication January 17, 2001. Accepted for publication September 12, 2001.
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,
CD28+/+ mice; 
, CD28-/- mice; 1st,
infection of naive mice; 2nd, secondary infection; ND, all naive mice
died 2–3 days after infection with 105
Listeria.
