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,
*
Immunology Graduate Program,
Center for Immunology, and
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas Texas 75235; and
§
Unité d’Immunité Cellulaire Antivirale, Institut Pasteur, Paris, 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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by NK cells has been shown to be crucial for
activating the bactericidal mechanisms of macrophages (3, 4). While the
innate immune response is efficient at limiting the initial spread of
infection, rapid clearance of Listeria depends on T
cell-mediated immunity (5). Both CD4+ and CD8+
T cells specific for LM Ags expand during the course of infection (6).
Although CD4+ T cells are capable of eliciting a
Listeria-specific delayed-type hypersensitivity response,
long term protective antilisterial immunity mainly resides within the
CD8+ T cell population (6, 7).
In addition to the presentation of Listeria Ags by MHC class
Ia proteins, nonclassical MHC class Ib molecules also present peptides
to antilisterial CTL. For example, early studies demonstrated the
generation of CD8+ T cells following LM infection that were
capable of providing short term antilisterial immunity upon adoptive
transfer into allogeneic strains of mice (8, 9). At first termed MHC
unrestricted, it is now understood that these effector cells recognize
Ags presented by nonclassical class I molecules. Class Ib MHC
molecules, encoded by the Q, T, and M regions, exhibit limited
polymorphism compared with their class Ia counterparts and are
therefore often shared among various strains of inbred mice. Three
LM-derived peptides have been identified that are presented to
CD8+ 
ß T cells by the class Ib molecule M3 (10, 11, 12). A
unique feature of M3 is its higher binding affinity for peptides
beginning with N-formyl methionine (13). Prokaryotic organisms initiate
protein synthesis with formylated methionine, which suggests that M3
has evolved to provide a specialized role in antimicrobial immunity.
The T-region-encoded class Ib molecule Qa1b has also been
shown to function as a restriction element for LM-specific CTL (14, 15). While the LM-derived Ag(s) presented to CD8+ T cells
by Qa1b has yet to be identified, a previous study
demonstrated that this process is TAP dependent (15), and thus a
peptide is most likely recognized.
MHC class Ib-restricted CTL are induced in vivo following a sublethal infection with LM (15). The lytic activity of these effectors expands and contracts with kinetics similar to those observed for class Ia-restricted antilisterial CTL. Despite these observations, the relative significance of class Ia vs class Ib presentation in generating antilisterial immunity has not been defined. In this study we infected mice lacking MHC class Ia molecules (H2 Kb-/- Db-/-) with LM to assess whether MHC class Ib Ag presentation plays a significant role in generating a CD8+ T cell response to this pathogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, TAP.1-/-, BALB/c, and Kb-/- Db-/- mice were bred and maintained in animal colonies at the University of Texas Southwestern Medical Center (Dallas, TX). Kb-/- Db-/- double-knockout mice have genomic deletions of the MHC class Ia genes Kb and Db and were generated from embryonic stem cells of strain 129/Ola (H2bc) as previously described (16). The knockout haplotype has been backcrossed onto the C57BL/6 (B6) background three times, and homozygotes were selected after intercrossing. Because the strain thus derives at least 87.5% of its genes from B6, B6 was the most suitable wild-type control strain for the experiments reported here. The Mhc of 129, and thus of the knockout mice, is considered identical with H2b of B6 in the class Ia, class II, and class III regions (the classical H2 complex); although 129 mice do differ from B6 in the H2-T region (reviewed in 17), they express the same forms of Qa1 (17, 18) and M3 (19), and no difference was noted between 129 and B6 in the H2-M region (20).
Bacteria
LM 10403 serotype 1 was provided by H. G. A. Bouwer (Veterans Affairs Medical Center, Portland, OR). Bacteria were grown on brain-heart infusion agar plates (BHI; Difco Laboratories, Detroit, MI), and virulent stocks were maintained by repeated passage through C57BL/6 mice. The LD50 dose for B6 mice is approximately 2 x 104 bacteria. To generate HKLM, log phase cultures of bacteria grown in BHI broth were washed twice with PBS and incubated for 1 h at 75°C. Bacterial numbers were determined by plating dilutions of viable bacteria on BHI agar plates and incubating for 24 h at 37°C. Colony counts were used to calculate the CFU per milliliter.
Cell lines and reagents
The J774 macrophage line (H2d) was kindly provided by H. G. A. Bouwer and is maintained in DMEM (Life Technologies, Gaithersburg, MD). HeLa cells and the Qa1b-expressing transfectant have been previously described (15) and are maintained in s-minimum essential medium (Life Technologies). CM3 cells are an M3wt transfectant of the B10.CAS2 fibroblast line (H2w17, M3cas) and were maintained in RPMI 1640 medium (Life Technologies). TAP.2-deficient RMA-S cells (H2b), Ltk cells (H2k), and Ltk transfectants expressing Kb, Db, and Qa1b were maintained in RPMI medium. All medium was supplemented with 10% FCS, 25 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 20 µM 2-ME. Cells were grown at 37°C in humidified air containing 7% CO2. For experiments in which cells would be infected with viable LM, antibiotic-free medium was used for passage.
Antibodies
For flow cytometric analysis, anti-CD4-PE (RM4-5),
anti-CD8-FITC (53-6.7), and anti-TCRß-biotin (H57-597) Abs
and streptavidin-PE were purchased from PharMingen (San Diego, CA).
Spleen cells (5 x 105) were stained for 20 min at
4°C and washed twice with PBS before analysis on a FACScan flow
cytometer (Becton Dickinson, Mountain View, CA).
For in vitro T cell depletions, anti-CD4 (2B6, rat IgM) and anti-CD8 (3.155, rat IgM) mAbs were semipurified from hybridoma-cultured medium by 50% ammonium sulfate precipitation and dialyzed three times against PBS.
Peptides
Peptide synthesis was performed by automated solid phase techniques using standard F-moc chemistry on a Rainin Symphony peptide synthesizer (Rainin, Woburn, MA). The homogeneity of each peptide was determined by reverse phase HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Stock solutions (2 mM) were prepared by dissolving lyophilized peptide in DMSO (Sigma, St. Louis, MO).
Generation of LM-specific CTL
For LM-specific CTL induction, mice were injected with approximately 2 x 103 bacteria in 200 µl of PBS via the tail vein. Six days following infection, spleen cells from these animals were cultured at 2 x 106/ml in a Corning T75 flask with 50 ml of complete RPMI medium containing 1 µg/ml Con A (Sigma). Cultures were maintained at 37°C in 7% CO2 humidified air for 72 h. These cells were then used as effectors in a 51Cr release CTL assay.
LM-specific CTL assay
Target cells (1–1.5 x 105) were plated in 48-well tissue culture plates 18 h before the assay in 0.5 ml of antibiotic-free medium. Log-phase cultures of LM grown in BHI broth were harvested and washed twice with PBS. Target cell monolayers were infected at a multiplicity of infection of 5:1 for J774 cells and 100:1 for HeLa, HeLa:Qa1b, B10.CAS2, and CM3 cells. Control cells received no bacteria. After 60–90 min, the target cell monolayers were washed twice with warm PBS, and 250 µl of medium containing 40 µg/ml gentamicin sulfate (Life Technologies) was added. Three hours after initiation of infection, 10 µl of 51Cr was added to each well and incubated for 1 h at 37°C. Target cell monolayers were then washed twice with PBS and covered in 250 µl of medium with gentamicin. Effector cells were added in 250 µl of medium. In certain experiments, J774 cells were incubated with HKLM (100:1) for 8 h at 37°C before effector addition. Spontaneous and maximum release wells received 250 µl of medium and 1% SDS, respectively. The assays were terminated 4–5 h later when 250 µl of culture supernatant was harvested and counted on a Micromedic ME plus gamma counter (Micromedic, Horsham, PA). The percent specific lysis was calculated as 100 x (experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm). Data presented are the means of duplicate wells.
In vivo LM CFU analysis
Mice were infected with 
2 x 103 LM in 200
µl of PBS via the tail vein. On days 3, 4, 7, 10, and 14 following
infection, the spleens and livers were removed and homogenized in
sterile water using a glass Dounce tissue grinder (Kontes, Vineland,
NJ). Serial 10-fold dilutions of the homogenate were plated in
triplicate on BHI agar plates and were incubated for 24 h at
37°C. Colony counts were averaged and corrected for dilution to yield
the LM CFU per organ. The CFU limit of detection is 50 for spleen and
100 for liver.
Adoptive transfer of antilisterial immunity
LM immune donor spleen cells were isolated from mice immunized 6 days previously with 2 x 103 LM and were divided into three groups. Two donor spleen groups were incubated (1 x 107/ml) for 30 min at 4°C with a 1/10 dilution of either 3.155 (anti-CD8) or 2B6 (anti-CD4) mAb. Cells were then washed twice and incubated (1 x 107/ml) with 10% rabbit complement (Pel-Freez, Brown Deer, WI) in a 37°C water bath for 30 min. Nondepleted LM immune and naive donor spleen groups were treated with complement only. Viable cells were recovered by centrifugation over a Ficoll gradient (Pharmacia, Piscataway, NJ) and subsequently washed twice with PBS. Naive syngeneic host animals received an i.v. injection of 5 x 107 donor spleen cells in 200 µl. Thirty minutes following cell transfer, mice were challenged i.v. with 4 x 104 LM. Three days after infection, spleens were removed from host animals, and the LM CFU per organ was calculated as previously described. Data are presented as the mean log10 CFU per spleen of three mice per donor cell group.
Generation of alloreactive CTL
Standard 5-day mixed lymphocyte cultures were established for the generation of alloreactive CTL. Irradiated stimulator spleen cells were obtained from female C57BL/6 (H2b) and BALB/c (H2d) mice. Stimulators were cultured in complete RPMI medium at 5 x 106/well in a 24-well tissue culture dish. Female Kb-/- Db-/- or C57BL/6 responder spleen cells were added at 5 x 106/well. Cultures were maintained at 37°C in 7% CO2 for 5 days.
Alloreactive and LM peptide-specific CTL assays
RMA-S cells were cultured with 10 µM peptide or an equivalent volume of DMSO for 14 h at 25°C before assay. Peptide-sensitized RMA-S cells (2 x 106) were labeled with 51Cr for 1.5 h at 25°C. All other target cells were labeled for 1 h at 37°C. Cells were then washed twice and added to a 96-well round-bottom plate at 5 x 103/well in 100 µl of complete RPMI medium. Titrations of effector cells were added in 100 µl of medium, and the assays were incubated for 4 h at 37°C. The supernatants (100 µl) were harvested, and the percent specific lysis was determined as described above. Data presented are the means of triplicate wells.
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Results |
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Naive Kb-/- Db-/- mice have 2%
CD8+ ß T cells in their spleens (Fig. 1
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population increases in response to an intracellular bacterial
infection, we injected Kb-/- Db-/- and B6
mice with a sublethal dose of LM. Six days following infection, spleen
cells were harvested, and the CD4+ and CD8+ T
cell populations were compared with those of naive animals. A three- to
fourfold increase in splenic CD8+ T cells occurred in
Kb-/- Db-/- mice following infection,
whereas no change in the CD4+ population was detected (Fig. 1). By contrast, no significant alteration in the percentage of
CD4+ or CD8+ T cells was observed in B6 mice
following LM infection.
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To determine whether the CD8+ T cell population
observed in Kb-/- Db-/- mice contained CTL
specific for LM, spleen cells from mice infected 6 days previously were
stimulated with Con A for 3 days and were used as effectors in a
51Cr release assay. The J774 macrophage line was
specifically lysed by Kb-/- Db-/- effectors
only upon infection with viable LM (Fig. 2A). The level of target cell
killing was comparable to that by B6 antilisterial CTL, whereas no
specific lysis was observed from naive B6 and Kb-/-
Db-/- (data not shown) effectors. The depletion of
CD8+ T cells from Con A cultured effectors before assay
resulted in the loss of CTL activity (data not shown). Because J774
(H2d) expresses allogeneic MHC class Ia, this suggests that
the CTL activity observed is MHC class Ib restricted.
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The MHC class Ib molecules M3 and Qa1b have previously
been shown to present LM Ags to CTL (15, 21). To examine whether
Kb-/- Db-/- antilisterial CTL were
restricted by either of these class Ib MHC molecules, transfected cell
lines expressing M3 and Qa1b were used as targets in a CTL
assay. Both B6 and Kb-/- Db-/- LM immune
effectors specifically lysed CM3 targets (H2w17,
M3wt) upon infection with LM, whereas the parental B10.CAS2
fibroblast line (H2w17, M3cas) was not lysed
(Fig. 2
B). These effector populations also lysed infected
HeLa cells expressing Qa1b, whereas parental HeLa cells
were not lysed (Fig. 2C). Effectors from a naive B6 or
Kb-/- Db-/- (data not shown) mouse were
incapable of lysing either target cell line upon LM infection. HeLa
cells transfected to express the nonclassical class I molecules CD1 and
Qa2 were also used as LM-infected targets in this assay. Neither cell
line was recognized by B6 or Kb-/- Db-/-
antilisterial CTL effectors (data not shown). These data demonstrate
that Kb-/- Db-/- CTL can recognize LM Ags
presented by the class Ib molecules M3 and Qa1b.
CTL recognition of Listeria Ags presented by M3
We examined the ability of B6 and Kb-/-
Db-/- antilisterial CTL to recognize and lyse target
cells sensitized with three LM-derived peptides previously demonstrated
to bind M3 (10, 11, 12). TAP-deficient RMA-S cells were incubated in the
presence or the absence of 10 µM peptide for 14 h at 25°C and
used as targets in a 51Cr release assay. RMA-S cells pulsed
with ND1, an N-formylated mitochondrium-derived peptide,
were used as a negative control. Both B6 (Fig. 3A) and Kb-/-
Db-/- (Fig. 3B) antilisterial CTL specifically
lysed target cells pulsed with the three LM peptides, whereas RMA-S
cells incubated without peptide or with the M3-restricted ND1 peptide
were not recognized. Both effector populations exhibited the highest
CTL activity against RMA-S cells pulsed with f-MIGWII, a peptide
derived from the LM LemA protein (11). B6 effectors lysed
f-MIVTLF-pulsed target cells at a higher level than f-MIVIL-pulsed
cells, while CTL activity against these peptides using
Kb-/- Db-/- effectors was relatively
equivalent. No target cell lysis was observed in the presence of naive
B6 effectors (Fig. 3C).
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Entry of LM Ags into the class I presentation pathway is believed
to require viable LM that are capable of lysing the phagosome and
entering the host cell cytosol (22, 23). Yet previous studies have
demonstrated that phagocytic cells pulsed with preparations of HKLM,
which do not gain access to the host cell cytosol, are capable of
presenting Ag to M3 restricted CTL clones in vitro (24, 25). Having
demonstrated the presence of polyclonal M3-restricted antilisterial CTL
in B6 and Kb-/- Db-/- effector populations,
we wanted to determine whether target cell recognition could occur in
the presence of HKLM or required infection with viable bacteria. J774
macrophage cells incubated with HKLM for 8 h at 37°C or infected
with virulent LM for 4 h were used as targets in a
51Cr release assay. Both B6 (Fig. 4A) and Kb-/-
Db-/- (Fig. 4B) antilisterial CTL, but not
naive B6 effectors (Fig. 4C), efficiently recognized and
lysed target cells infected with viable LM. CTL activity against J774
targets pulsed with HKLM was not any greater than that observed against
untreated J774 cells, demonstrating that this is not a relevant pathway
for class Ib presentation of LM Ags. Extending the time of target cell
incubation with HKLM to 18 h did not enhance CTL recognition (data
not shown).
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We next examined whether the immune response elicited in
Kb-/- Db-/- animals was efficient in
clearing an in vivo LM infection from spleen and liver. For these
experiments we also used TAP.1-/- mice, which express
very low levels of class Ia and, presumably, class Ib molecules and
have approximately 1% CD8+ T cells in the spleen (26). B6,
Kb-/- Db-/-, and TAP.1-/-
mice were infected with a sublethal dose of LM. At various times
following infection, spleens and livers were harvested from these
animals, and the LM CFU per organ was measured. Kb-/-
Db-/- mice clear LM from the spleen with kinetics similar
to those observed in B6 mice, which induce both class Ia- and class
Ib-restricted CTL (Fig. 5). No B6 mice
had detectable LM on day 10, and only one of seven Kb-/-
Db-/- mice showed CFU in its spleen at that time. By
contrast, TAP.1-/- mice still showed evidence of
infection on day 14. A similar bacterial clearance rate was observed in
the livers of these animals (data not shown).
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We conducted adoptive transfer experiments to further demonstrate
that CD8+ T cells in Kb-/-
Db-/- mice were involved in protection against LM
infection. LM immune spleen cells from B6 or Kb-/-
Db-/- mice infected 6 days previously with a low dose of
LM were left untreated or were depleted of CD4+ T cells or
CD8+ T cells in vitro. Donor cells (5 x
107) were infused into naive syngeneic recipients, and 30
min later these animals were challenged with 4 x 104
LM. Three days following LM infection the spleens were harvested, and
the LM CFU per organ was determined. As shown in Table I, transfer of LM immune spleen cells
resulted in 5.6 log10 protection in B6 recipient mice and
3.4 log10 protection in Kb-/-
Db-/- mice when compared with control animals that
received naive donor spleen cells. Both B6 and Kb-/-
Db-/- LM immune donor cells depleted of CD4+
T cells were still capable of conferring protection in naive host
animals, whereas almost all protection was lost when donor cells were
depleted of CD8+ T cells. These data demonstrate that, as
in B6 mice, CD8+ T cells in Kb-/-
Db-/- animals play an important role in the protective
immune response against LM infection.
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We tested whether spleen cells from Kb-/-
Db-/- mice could generate a CD8+ T cell
response against MHC class Ia Ags in an in vitro primary MLC.
Kb-/- Db-/- and B6 responder spleen cells
were cultured in the presence of irradiated stimulator cells from
BALB/c (H2d) or B6 (H2b) mice. After 5 days of
culture, we observed a >20-fold increase in CD8+ T cells
in both B6 and Kb-/- Db-/- anti-BALB/c
MLC and Kb-/- Db-/- anti-B6 MLC, but
not in B6 anti-B6 MLC (data not shown). These effector populations
were then tested for their cytolytic activity against P815
(H2d) and RMA (H2b) targets to determine
whether alloreactive CTL were present. Spleen cells from B6 and
Kb-/- Db-/- mice were cultured with Con A
and used as negative control effectors. Both B6 and Kb-/-
Db-/- anti-BALB/c MLC generate CTL capable of lysing
P815 target cells (Fig. 6A).
Kb-/- Db-/- anti-B6 MLC effectors lyse
RMA cells, whereas B6 anti-B6 MLC effectors fail to lyse the
syngeneic target (Fig. 6B). No CTL activity was observed in
these experiments using Con A-cultured effectors. To assess which MHC
class Ia proteins were targets for Kb-/-
Db-/- anti-B6 CTL, Ltk (H2k) cells
expressing Kb or Db were used as targets. L
cells expressing both Kb (Fig. 6C) and
Db (Fig. 5D) were specifically lysed by these
CTL, whereas parental L cells were not recognized (Fig. 6E).
In contrast to a previous report (27), these experiments demonstrate
that unprimed CD8+ T cells from Kb-/-
Db-/- mice are capable of generating a response against
MHC class Ia Ags from the H2d, H2b, and
H2k (data not shown) haplotypes following primary in vitro
culture.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following a sublethal LM infection, we observe an increase of
CD8+ ß T cells in the spleen of Kb-/-
Db-/- mice, which are capable of lysing infected target
cells in vitro. We demonstrate that these antilisterial CTL recognize
LM Ags presented by the class Ib molecules, Qa1b and M3.
Previous studies that have analyzed the three LM-derived antigenic
peptides presented by M3 used CTL lines or clones propagated in vitro,
while the in vivo response to these Ags has remained undefined. In this
study we have compared the CTL activity against the M3-restricted
peptides using polyclonal stimulated spleen cells from B6 and
Kb-/- Db-/- mice infected with LM. While
all three LM peptides were recognized by these effector cells, both B6
and Kb-/- Db-/- antilisterial CTL exhibited
the highest lytic activity against target cells pulsed with f-MIGWII.
The fact that CTL recognition of these epitopes titrated further using
Kb-/- Db-/- effector cells (6:1), compared
with B6 effector cells (50:1), may indicate that a higher frequency of
antilisterial CTL are restricted by M3 in these animals. Previous
evidence has suggested that the M3 class Ib molecule can present LM Ags
in a nonconventional manner. Data demonstrating that J774 macrophage
cells pulsed with HKLM can present Ag to M3-restricted CTL clones has
led to the hypothesis that M3 can associate with peptides outside the
endoplasmic reticulum, perhaps binding Ag within phagocytic vacuoles
analogous to the class I-like molecule CD1 (24). Our results, however,
demonstrate that J774 cells infected with viable LM, and not cells
pulsed with HKLM, are capable of presenting LM Ags to class
Ib-restricted antilisterial CTL from B6 and Kb-/-
Db-/- mice. These conflicting results can be attributed
to the effector population used for analysis. Previous studies
demonstrating recognition of HKLM Ags have used peptide-specific CTL
clones or purified CD8+ T cell lines that were maintained
in vitro with high concentrations of HKLM, most likely resulting in the
selection of high affinity CTL that were capable of responding to low
levels of Ag (8, 24, 25). Our experiments are conducted using
polyclonal stimulated T cells from an immunized animal, thus more
accurately reflecting the CTL specificities generated during an in vivo
infection. While one report has demonstrated that IL-2-cultured
CD8+ T cells from LM-immune animals could lyse macrophages
pulsed with HKLM in a class Ib-restricted manner (9), we did not
observe such recognition. Our findings demonstrate that, as for class
Ia molecules, efficient presentation of LM Ags by M3 and other class Ib
molecules requires infection with viable bacteria.
While the antigenic peptide presented by Qa1b is unknown, it is most likely derived from a protein secreted by LM in the host cell cytosol, like all other LM class I epitopes characterized to date (29). Qa1 and its human homologue HLA-E are unique class I molecules in that they preferentially bind conserved peptides derived from the leader sequence of MHC class Ia molecules (30, 31). In fact, the peptide binding groove of Qa1b is almost exclusively occupied by a single high affinity peptide derived from the signal sequence of D region molecules (32, 33). While it has been hypothesized that Qa1 functions in the regulation of NK cells (30), the possibility exists that the restrictive peptide binding groove has evolved for the binding and presentation of conserved pathogen-derived peptides, perhaps from signal sequences. Whether the absence of the high affinity Db leader peptide in Kb-/- Db-/- mice facilitates loading of the LM-derived epitope, therefore eliciting a stronger Qa1b-restricted CTL response in these animals, is not currently known. The ratio of M3- vs Qa1b-restricted antilisterial CTL in Kb-/- Db-/- mice and whether other class Ib proteins play a role in generating immunity to LM are also questions of interest. Our studies to date have excluded the presentation of LM Ags by CD1 and Qa2.
Our in vitro data suggest that class Ib presentation of LM Ags is
relevant for eliciting CD8+ T cell immunity in
Kb-/- Db-/- animals. These data are further
supported by our in vivo analysis, which demonstrates that
Kb-/- Db-/- mice clear LM from spleen and
liver with the same kinetics as wild-type B6 mice and more rapidly than
TAP.1-/- animals, which exhibit low expression of class I
molecules. Rapid reduction of LM CFU in the liver of
Kb-/- Db-/- mice also corroborates prior
evidence that infected hepatocytes can be lysed in a class
Ib-restricted manner (34). It is not currently understood whether the
lack of MHC class Ia proteins alters the innate immune response to LM
infection. However, it is of interest to note that in the early phase
of infection (day 3), Kb-/- Db-/- mice
consistently have lower LM CFU in the spleen than B6 or
TAP.1-/- animals (Fig. 5). The activities of macrophages,
NK cells, neutrophils, and 
T cells following infection of
Kb-/- Db-/- mice are currently being
investigated. We have further defined the role of CD8+ T
cells in Kb-/- Db-/- mice by examining the
ability of LM immune spleen cells to transfer protection to naive
hosts. The adoptive transfer of Kb-/- Db-/-
spleen cells depleted of CD4+ T cells protected recipients
from subsequent LM challenge, whereas the transfer of
CD8+-depleted cells did not. Together with our in vitro
demonstration of M3- and Qa1b-restricted LM-specific CTL,
these data indicate that class Ib molecules present LM Ags to
CD8+ T cells, which are probably responsible for generating
sterilizing immunity following in vivo infection. A study comparing the
in vivo responses of class Ia- and class Ib-restricted CD8+
T cells has demonstrated that the CTL activities of the two populations
expand and contract with similar kinetics, correlating with the
clearance of bacterial infection (15). Despite these findings, the
relative frequency of antilisterial CTL restricted by class Ia vs class
Ib in a wild-type animal has not been defined. A recent report has
analyzed four different class Ia-restricted CTL populations from BALB/c
mice that recognize LM Ags presented by Kd (35). At the
peak antilisterial CTL response 7 days postinfection, these CTL groups
together represented only approximately 2% of all splenic
CD8+ T cells. The three- to fourfold increase in
CD8+ T cells in Kb-/- Db-/-
mice 6 days after LM challenge suggests that class Ib-restricted CTL
are capable of expanding in response to bacterial infection, although
the possibility that these cells are recruited from another site within
these animals cannot be excluded. Whether the class Ib-restricted CTL
population exhibits the same expansion in wild-type mice remains to be
determined.
How the peripheral CD8+ T cell repertoire is generated in Kb-/- Db-/- mice is not known. Our data demonstrate that in the absence of MHC class Ia molecules, these mice exhibit a substantial reduction in peripheral CD8+ lymphocytes compared with wild-type animals. The ability of splenic CD8+ T cells from Kb-/- Db-/- mice to mount a vigorous alloresponse against polymorphic MHC class Ia molecules following primary in vitro culture suggests that this population has undergone selection on structurally similar class Ib MHC. However, it is also possible that recognition of MHC class I molecules is inherent in the germline of TCRs independent of positive and negative selection (36). If the former possibility is correct, the drastic reduction in peripheral CD8+ T lymphocytes indicates that only a minor population of thymocytes is capable of being selected on class Ib molecules. If the repertoire of mature CD8+ T cells in Kb-/- Db-/- mice is limited, it is surprising that we observed a three- to fourfold increase in splenic CD8+ T cells 6 days following LM inoculation, but found no significant change in B6 mice. This difference may reflect that the percentage of naive precursor CD8+ lymphocytes in the T cell pool capable of recognizing LM antigens is substantially higher in these animals than in wild-type mice.
We have also conducted experiments to determine whether Kb-/- Db-/- mice are capable of generating a CTL response against viral pathogens. Our results demonstrate that infection of these animals with vaccinia virus, vasicular stomatitis virus (data not shown), and lymphocytic choriomeningitis virus (16) fails to elicit CD8+ T cell immunity. The failure to detect viral Ags due to a limited repertoire of TCRs expressed by the peripheral CD8+ population could explain this lack of CTL activation. Whether any virus-derived peptides are capable of binding to class Ib molecules for cell surface presentation is also not known. While M3 can bind a nonformylated peptide derived from the influenza hemagglutinin protein for presentation to CD8+ T cells in vitro (37), the presentation of this peptide in vivo for activation of a CTL response has yet to be demonstrated.
In summary, we have shown that in the absence of MHC class Ia molecules, antilisterial CTL immunity can be efficiently generated through MHC class Ib Ag presentation. It will be of interest to determine whether the limited polymorphic nature of class Ib proteins can be targeted for peptide vaccination strategies, eliciting CD8+ T cell immunity to bacterial Ags in various strains of inbred mice that express disparate MHC class Ia proteins.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James Forman, Center for Immunology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75235-9093. E-mail address:
3 Abbreviations used in this paper: LM, Listeria monocytogenes; BHI, brain-heart infusion; HKLM, heat-killed L. monocytogenes.
Received for publication December 8, 1998. Accepted for publication February 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are costimulators of interferon- production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725. interferon production by natural killer cells is important in defense against murine listeriosis. Infect. Immun. 59:2892.This article has been cited by other articles:
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  A. Tvinnereim and B. Wizel CD8+ T Cell Protective Immunity against Chlamydia pneumoniae Includes an H2-M3-Restricted Response That Is Largely CD4+ T Cell-Independent J. Immunol., September 15, 2007; 179(6): 3947 - 3957. [Abstract] [Full Text] [PDF]
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T. van Hall, S. Laban, D. Koppers-Lalic, J. Koch, C. Precup, P. Asmawidjaja, R. Offringa, and E. J. H. J. Wiertz The Varicellovirus-Encoded TAP Inhibitor UL49.5 Regulates the Presentation of CTL Epitopes by Qa-1b1 J. Immunol., January 15, 2007; 178(2): 657 - 662. [Abstract] [Full Text] [PDF]
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M. T. Chow, S. Dhanji, J. Cross, P. Johnson, and H.-S. Teh H2-M3-Restricted T Cells Participate in the Priming of Antigen-Specific CD4+ T Cells J. Immunol., October 15, 2006; 177(8): 5098 - 5104. [Abstract] [Full Text] [PDF]
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 O. Boyman, J.-H. Cho, J. T. Tan, C. D. Surh, and J. Sprent A major histocompatibility complex class I-dependent subset of memory phenotype CD8+ cells J. Exp. Med., July 10, 2006; 203(7): 1817 - 1825. [Abstract] [Full Text] [PDF]
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S. E.F. D'Orazio, C. A. Shaw, and M. N. Starnbach H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes J. Exp. Med., February 21, 2006; 203(2): 383 - 391. [Abstract] [Full Text] [PDF]
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H. Xu, T. Chun, H.-J. Choi, B. Wang, and C.-R. Wang Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense J. Exp. Med., February 21, 2006; 203(2): 449 - 459. [Abstract] [Full Text] [PDF]
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J. Su, R. E. Berg, S. Murray, and J. Forman Thymus-Dependent Memory Phenotype CD8 T Cells in Naive B6.H-2Kb-/-Db-/- Animals Mediate an Antigen-Specific Response against Listeria monocytogenes J. Immunol., November 15, 2005; 175(10): 6450 - 6457. [Abstract] [Full Text] [PDF]
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M. Chen, P. Tabaczewski, S. M. Truscott, L. Van Kaer, and I. Stroynowski Hepatocytes Express Abundant Surface Class I MHC and Efficiently Use Transporter Associated with Antigen Processing, Tapasin, and Low Molecular Weight Polypeptide Proteasome Subunit Components of Antigen Processing and Presentation Pathway J. Immunol., July 15, 2005; 175(2): 1047 - 1055. [Abstract] [Full Text] [PDF]
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L. M. Reed-Loisel, B. A. Sullivan, O. Laur, and P. E. Jensen An MHC Class Ib-Restricted TCR That Cross-Reacts with an MHC Class Ia Molecule J. Immunol., June 15, 2005; 174(12): 7746 - 7752. [Abstract] [Full Text] [PDF]
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B. A. Sullivan, L. M. Reed-Loisel, G. J. Kersh, and P. E. Jensen Homeostatic Proliferation of a Qa-1b-Restricted T Cell: A Distinction between the Ligands Required for Positive Selection and for Proliferation in Lymphopenic Hosts J. Immunol., November 15, 2004; 173(10): 6065 - 6071. [Abstract] [Full Text] [PDF]
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 S. E. F. D'Orazio, M. Velasquez, N. R. Roan, O. Naveiras-Torres, and M. N. Starnbach The Listeria monocytogenes lemA Gene Product Is Not Required for Intracellular Infection or To Activate fMIGWII-Specific T Cells Infect. Immun., December 1, 2003; 71(12): 6721 - 6727. [Abstract] [Full Text] [PDF]
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A. Ploss, G. Lauvau, B. Contos, K. M. Kerksiek, P. D. Guirnalda, I. Leiner, L. L. Lenz, M. J. Bevan, and E. G. Pamer Promiscuity of MHC Class Ib-Restricted T Cell Responses J. Immunol., December 1, 2003; 171(11): 5948 - 5955. [Abstract] [Full Text] [PDF]
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 L. Cao, C. A. Hudson, and D. A. Lawrence Immune Changes during Acute Cold/Restraint Stress-Induced Inhibition of Host Resistance to Listeria Toxicol. Sci., August 1, 2003; 74(2): 325 - 334. [Abstract] [Full Text] [PDF]
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C. K. Doyle, B. K. Davis, R. G. Cook, R. R. Rich, and J. R. Rodgers Hyperconservation of the N-Formyl Peptide Binding Site of M3: Evidence that M3 Is an Old Eutherian Molecule with Conserved Recognition of a Pathogen-Associated Molecular Pattern J. Immunol., July 15, 2003; 171(2): 836 - 844. [Abstract] [Full Text] [PDF]
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S. E. F. D'Orazio, D. G. Halme, H. L. Ploegh, and M. N. Starnbach Class Ia MHC-Deficient BALB/c Mice Generate CD8+ T Cell-Mediated Protective Immunity Against Listeria monocytogenes Infection J. Immunol., July 1, 2003; 171(1): 291 - 298. [Abstract] [Full Text] [PDF]
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Z. Kurepa, J. Su, and J. Forman Memory Phenotype of CD8+ T Cells in MHC Class Ia-Deficient Mice J. Immunol., June 1, 2003; 170(11): 5414 - 5420. [Abstract] [Full Text] [PDF]
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A. Davies, S. Kalb, B. Liang, C. J. Aldrich, F. A. Lemonnier, H. Jiang, R. Cotter, and M. J. Soloski A Peptide from Heat Shock Protein 60 Is the Dominant Peptide Bound To Qa-1 in the Absence of the MHC Class Ia Leader Sequence Peptide Qdm J. Immunol., May 15, 2003; 170(10): 5027 - 5033. [Abstract] [Full Text] [PDF]
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K. B. Urdahl, D. Liggitt, and M. J. Bevan CD8+ T Cells Accumulate in the Lungs of Mycobacterium tuberculosis-Infected Kb-/-Db-/- Mice, But Provide Minimal Protection J. Immunol., February 15, 2003; 170(4): 1987 - 1994. [Abstract] [Full Text] [PDF]
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E. Y. Chiang, M. Henson, and I. Stroynowski The Nonclassical Major Histocompatibility Complex Molecule Qa-2 Protects Tumor Cells from NK Cell- and Lymphokine-Activated Killer Cell-Mediated Cytolysis J. Immunol., March 1, 2002; 168(5): 2200 - 2211. [Abstract] [Full Text] [PDF]
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 J. M. Antonini, J. R. Roberts, R. W. Clarke, H.-M. Yang, M. W. Barger, J. Y. C. Ma, and D. N. Weissman Effect of Age on Respiratory Defense Mechanisms : Pulmonary Bacterial Clearance in Fischer 344 Rats After Intratracheal Instillation of Listeria monocytogenes Chest, July 1, 2001; 120(1): 240 - 249. [Abstract] [Full Text] [PDF]
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G. Lauvau and E. G. Pamer CD8 T Cell Detection of Bacterial Infection: Sniffing for Formyl Peptides Derived from Mycobacterium tuberculosis J. Exp. Med., May 21, 2001; 193(10): F35 - F40. [Full Text] [PDF]
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H. G. A. Bouwer, R. A. Barry, and D. J. Hinrichs Lack of Expansion of Major Histocompatibility Complex Class Ib-Restricted Effector Cells following Recovery from Secondary Infection with the Intracellular Pathogen Listeria monocytogenes Infect. Immun., April 1, 2001; 69(4): 2286 - 2292. [Abstract] [Full Text] [PDF]
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A. Bai, C. J. Aldrich, and J. Forman Factors Controlling the Trafficking and Processing of a Leader-Derived Peptide Presented by Qa-1 J. Immunol., December 15, 2000; 165(12): 7025 - 7034. [Abstract] [Full Text] [PDF]
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M. S. Seaman, C.-R. Wang, and J. Forman MHC Class Ib-Restricted CTL Provide Protection Against Primary and Secondary Listeria monocytogenes Infection J. Immunol., November 1, 2000; 165(9): 5192 - 5201. [Abstract] [Full Text] [PDF]
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M. S. Rolph and S. H. E. Kaufmann Partially TAP-Independent Protection Against Listeria monocytogenes by H2-M3-Restricted CD8+ T Cells J. Immunol., October 15, 2000; 165(8): 4575 - 4580. [Abstract] [Full Text] [PDF]
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 M. J. Soloski, M. E. Szperka, A. Davies, and S. L. Wooden Host Immune Response to Intracellular Bacteria: A Role for MHC-Linked Class-Ib Antigen-Presenting Molecules Experimental Biology and Medicine, September 1, 2000; 224(4): 231 - 239. [Abstract] [Full Text]
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J. R. Kraft, R. E. Vance, J. Pohl, A. M. Martin, D. H. Raulet, and P. E. Jensen Analysis of Qa-1b Peptide Binding Specificity and the Capacity of CD94/NKG2A to Discriminate between Qa-1-Peptide Complexes J. Exp. Med., August 28, 2000; 192(5): 613 - 624. [Abstract] [Full Text] [PDF]
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 H. Jiang, N. S. Braunstein, B. Yu, R. Winchester, and L. Chess CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice PNAS, May 22, 2001; 98(11): 6301 - 6306. [Abstract] [Full Text] [PDF]
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), ND1 (f-MFFINVLTL; 
),
f-MIGWII (•), f-MIVIL (
), or f-MIVTLF (
) for 14 h at
25°C and then used as targets in a 51Cr release assay.
LM-infected B6 (A), LM-infected Kb-/-
Db-/- (B), and naive B6 (C)
effector cells were generated as described in Materials and
Methods. Results are expressed as the percent specific
51Cr release, and are the means of triplicate wells.
), B6 anti-B6 MLC (