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Infectious Diseases and
Immunobiology, Yale University School of Medicine, New Haven, CT 06520
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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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Upon cellular infection, L. monocytogenes escapes the phagosomal vacuole, entering the host cell cytosol where it divides and locomotes by polymerizing actin (11). Bacterial proteins secreted by L. monocytogenes into the host cell cytosol are degraded into peptides for presentation by MHC class I molecules to CD8 T lymphocytes (12, 13). Several L. monocytogenes-derived peptide epitopes that stimulate CD8 T cell responses during murine infection have been identified (14). The expansion and contraction of CD8 T cell responses specific for four different L. monocytogenes-derived epitopes were recently shown to be remarkably synchronous (15). Coordinate regulation of T cell responses to these different epitopes was surprising; previous studies had shown that the epitopes are present in infected cells in remarkably different amounts and have very different binding stabilities with the MHC class I molecule H2-Kd (12). This result suggested that the duration of T cell expansion following L. monocytogenes infection is not determined by the duration of in vivo Ag presentation.
The factors that determine the magnitude of T cell responses following viral or bacterial infection remain incompletely defined. A comfortable but unproven assumption is that the magnitude of T cell responses is determined by the amount of Ag and the duration of its presentation. Ample evidence, however, suggests that Ag quantity and stability do not determine the magnitude of T cell responses (15, 16, 17, 18). In the L. monocytogenes model, the immunodominant T cell response is specific for a peptide that is far less prevalent than two other peptides that elicit substantially smaller T cell responses (16, 19). Similarly, the correlation between Ag quantity and the size of the T cell response has been broken in studies of the murine T cell response to influenza virus infection (17).
In this report, we investigate the effect of severity and duration of in vivo infection on expansion of Ag-specific CD8 T cells specific for L. monocytogenes. We found that the rate and duration of in vivo T cell proliferation following priming is independent of the duration of infection or the quantity of Ag present in vivo. Our results suggest that pathogen-specific T lymphocytes are programmed during the first day of infection and subsequently undergo proliferation and differentiation into effector T cells without further calibration by the progressing inflammatory response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME). BALB/c Thy1.1-congenic mice were provided by Charles Surh (The Scripps Research Foundation, La Jolla, CA). L. monocytogenes strain 10403S was provided by Daniel Portnoy (University of California, Berkeley, CA).
Flow cytometry and tetramer generation
H2-Kd tetramers complexed with
listeriolysin O
(LLO)591–99
and p60449–457 were generated as
previously described (15). Epitope-specific T cell
populations were detected with PE-conjugated, tetrameric MHC-peptide
complexes and concurrently stained for other surface molecules using
directly conjugated mAbs as described previously (15).
Briefly, after blocking with unconjugated streptavidin (0.5 mg/ml;
Molecular Probes, Eugene, OR) and Fc block (PharMingen, San Diego, CA),
1–2 x 106 splenocytes were incubated in
FACS staining buffer (PBS (pH 7.45), 0.5% BSA, and 0.02% sodium
azide) for 1 h on ice in the presence of saturating concentrations
of tetramer reagents (0.25–0.5 mg/ml) and the various mAbs. Cells were
subsequently washed three times in staining buffer and then fixed in
1% paraformaldehyde/PBS (pH 7.45). Flow cytometry was performed using
a FACScalibur, and data were further analyzed with CellQuest software
(Becton Dickinson, Mountain View, CA). In some cases (see Fig. 7
), dead
cells were excluded by staining with ethidium monoazide bromide
(Molecular Probes) at a concentration of 1.25 mg/ml, added during
staining, with exposure to light during the last 10 min of staining.
The following mAbs were used (all obtained from PharMingen):
Cy-Chrome-conjugated anti-CD8
(clone 53-6.7), FITC-conjugated
anti-CD62L (clone MEL-14), and FITC-conjugated anti-CD4 (clone
H129.19).
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and TNF assays
Spleens were removed from naive BALB/c mice, L.
monocytogenes-infected mice that had received ampicillin for
48 h beginning 1 day after infection and infected mice that were
not treated with antibiotics. Spleens were macerated into ice-cold PBS
containing 0.01% Triton X-100 and centrifuged at 10,000 x
g for 10 min. The amounts of murine IFN- and TNF in the
spleen supernatants were determined by sandwich ELISA using OptEIA kits
from PharMingen, following the manufacturer’s protocols.
CTL assays
CD8 T cells were enriched from spleens by negative selection using magnetically activated cell sorting (MACS, Miltenyi, Germany) with anti-rat IgG microbeads and anti-CD4 (GK1.5) and anti-MHC class II (TIB120). Target cells were labeled with 51Cr, coated with 10-6 M LLO91–99, and incubated in the presence of enriched CD8 T cells at an E:T ratio of 33:1. After 4 h of incubation, the released 51Cr was quantified and expressed as the percent specific lysis, as previously described (20).
Generation of TCR-transgenic mice
VDJ regions of the TCR ß- and -chain genes were PCR
amplified from genomic DNA of CTL clone WP11.12, which is specific for
the L. monocytogenes- derived, subdominant epitope
p60449–457 (14). The ß-chain VDJ
region was amplified with plaque-forming units of polymerase
(Stratagene, La Jolla, CA) using the sense Vß8.2 primer
5'-CGCTCGAGGGAAGCATGGGCTCCAGGCTCTTCTTCGTG-3' and the antisense
Jß1.1 primer 5'-TCTTATCGATTGCACATCAGAATACAGATACTCG-3'. The resulting
fragment was digested with XhoI and ClaI and
cloned into the TCR ß cassette (44). The TCR -chain
VJ region was amplified using the sense V8 primer
5'-CGCCCGGGATGAAC-ATGCGTCCTGTCACCTGCTCAGTT-3' and the antisense
J20 primer 5'-GCGGCGGCCGTCCCTACTCTGTCCTTATGAG-3' that include
XmaI and NotI restriction sites, respectively.
This fragment was digested with XmaI and NotI and
cloned into the TCR cassette. Plasmids were screened by restriction
mapping for the presence of the complete TCR ß or gene. The TCR
ß gene insert was separated from the prokaryotic fragment of the
vector by digestion with KpnI and the TCR gene insert by
digestion with SalI. These DNA fragments were purified and
comicroinjected into fertilized oocytes of (C57BL6 x
SJL)F1 mice at the Yale transgenic mouse
facility. Founders were identified by PCR screening of DNA purified
from mouse tails and were bred for 10 generations onto the BALB/cJ
background.
Adoptive transfer of CFSE-labeled splenocytes
Splenocytes were isolated from WP11.12 TCR-transgenic mice and 1 x 107 cells were labeled in 1 ml of 5 µM CFSE in PBS at 37°C for 10 min. Cells were washed with RPMI 1640/10% FCS and resuspended in PBS for i.v. inoculation into recipient mice. Recipients were inoculated with 30 million CFSE-labeled splenocytes and infected 1 day later with 2000 L. monocytogenes or left uninfected.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This result is striking because the
severity of infection and the amount of Ag present in vivo are markedly
different in these settings (22). This result suggests
that neither the amount of Ag presented in vivo nor the severity of the
inflammatory response induced by these infections significantly
influences the rate at which LLO91–99-specific T
lymphocytes expand in vivo.
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A). Cultures of liver also
demonstrated complete elimination of viable bacteria (results not
shown). In contrast, untreated mice had viable L.
monocytogenes isolated from the spleen and liver for up to 7 days
following infection. Thus, ampicillin therapy rapidly eliminates
bacterial infection and dramatically alters the in vivo Ag load. To
determine whether the inflammatory response was also attenuated in
antibiotic-treated mice, we measured the amounts of TNF and IFN- in
spleens of mice that were treated for 48 h with ampicillin or left
untreated. As shown in Fig. 2, B and C, 48 h
after initiation of antibiotics (3 days following bacterial
inoculation), the amounts of TNF and IFN- in the spleen are
indistinguishable from uninfected control mice. In contrast, L.
monocytogenes-infected untreated mice have elevated levels of
these two cytokines. This result indicates that early antibiotic
treatment of mice infected with L. monocytogenes
down-regulates the in vivo inflammatory response in addition to
eliminating viable bacteria.
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).
Mice were divided into four groups that either received no antibiotics
or were treated with ampicillin at 0, 12, or 24 h after bacterial
inoculation, and the LLO91–99-specific T cell
response was measured 7 days after infection. Mice that received
antibiotics immediately following L. monocytogenes
inoculation had essentially undetectable
LLO91–99-specific T cell responses. Mice that
received ampicillin 12 h after infection mounted
LLO91–99-specific T cell responses of
intermediate size, whereas mice that received ampicillin 24 h
after infection had nearly normal
LLO91–99-specific T cell responses when compared
with untreated mice. These results indicate that the magnitude of the
LLO91–99-specific response correlates with the
duration of in vivo bacterial growth during the first 24 h after
infection.
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A). Although the
absolute number of LLO91–99-specific T cells is
lower when ampicillin is administered 24 h after L.
monocytogenes inoculation, reflecting the smaller spleen size in
antibiotic-treated mice, the peak frequency of the Ag-specific T cell
response occurs 7–9 days after infection in both cases. This finding
indicates that the kinetics of L. monocytogenes-specific T
cell expansion and contraction is neither dictated by the duration of
infection nor by the associated inflammatory response following the
first day of bacterial infection.
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production following Ag exposure and
direct ex vivo cytolytic activity (15, 16). To test
whether LLO91–99-specific CD8 T cells in mice
following early termination of infection have acquired effector
function, we assayed enriched CD8 T cells isolated from
ampicillin-treated and untreated mice for direct cytolytic activity on
LLO91–99-coated target cells. Fig. 4
B
demonstrates that the ex vivo cytolytic activity toward
LLO91–99-coated target cells was similar in mice
that had undergone attenuated infection and mice that underwent the
normal course of infection. Thus, prolonged infection and inflammation
are not necessary for L. monocytogenes-specific T cells to
acquire effector function.
To determine whether memory T cell generation was affected by
early termination of bacterial infection, we measured
LLO91–99-specific T cell populations 5 days
after reinfection of mice that had been immunized 1 mo earlier with
2000 L. monocytogenes followed by either no antibiotic
therapy or oral ampicillin at 0, 12, or 24 h after bacterial
inoculation (Fig. 5). All four groups of
mice survived a lethal challenge of 100,000 L.
monocytogenes, demonstrating that even mice immediately treated
with antibiotics following infection had acquired protective immunity.
The memory CD8 T cell response to LLO91–99 was
identical in mice that received either no antibiotics or ampicillin
12–24 h after infection (Fig. 5), indicating that progressive
bacterial infection during the CD8 T cell expansionary phase is not
required for memory generation. As might be expected, the markedly
diminished primary response to LLO91–99 in the
setting of immediate antibiotic therapy following infection resulted in
an attenuated memory response to this epitope.
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5 x 104, we
treated infected mice with ampicillin 24 h after inoculation with
either 10,000, 100,000, or 1,000,000 live L. monocytogenes
(Fig. 6). Remarkably, the magnitude of
the LLO91–99-specific T cell responses was
similar even though the doses of Ag differed by a factor of 100. Thus,
in agreement with the findings presented in Fig. 1, exceeding the
threshold for complete activation of available naive T cells does not
result in further increases in the magnitude of the T cell response.
Previous studies from our laboratory have shown that increasing the
presentation of epitope above the threshold required for T cell priming
does not enhance T cell responses (21). At the extreme
end, it has been shown that presentation of massive amounts of Ag
induces clonal T cell exhaustion (23). The amount of Ag
that is presented during murine infection with L.
monocytogenes most likely does not approach the amount that is
required to induce clonal exhaustion. Our results show that the size of
the T cell response is not significantly different following
inoculation with a nearly 1000-fold dose range of viable bacteria (see
Figs. 1 and 6), demonstrating a remarkable degree of independence
between T cell responses and in vivo Ag presentation and
infection-induced inflammation.
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). This
finding suggests that, during the first 24 h of infection, the
magnitude of the primary T cell response to L. monocytogenes
infection correlates with the duration of in vivo bacterial
replication. One explanation for this finding is that the number of
naive T cells that are primed during the first day of infection is time
dependent. This would suggest that 24 h of viable bacterial
infection are required for most naive T cells to come into contact with
APCs. An alternative explanation for differing T cell response
magnitudes in the setting of early antibiotic therapy is that the rate
or extent of T cell proliferation differs. Thus, T cell populations
primed by very short exposure to Ag, such as when antibiotic
administration begins 12 h after bacterial inoculation, may
undergo fewer in vivo divisions than T cells exposed to 24 h of
infection. To differentiate between these two possibilities, we
generated transgenic mice expressing the TCR for the L.
monocytogenes-derived, H2-Kd-restricted
p60449–457 epitope. In contrast to
LLO91–99, p60449–457 is a
subdominant epitope which elicits a nearly undetectable T cell response
following primary L. monocytogenes infection (15, 16). However, p60449–457 is generated
with great efficiency and is the most prevalent epitope presented by
H2-Kd in L. monocytogenes-infected
cells (19). Although p60449–457 is
highly prevalent in cells infected with live bacteria, it is rapidly
lost from infected cells because the epitope dissociates from
H2-Kd with a half-life of 1 h, whereas
LLO91–99 has a half-life that exceeds 6 h
(24). Thus, in the presence of antibiotics that
inhibit L. monocytogenes,
p60449–457 is rapidly lost
(24). Fig. 7 demonstrates
that TCR-transgenic mice contain a normal proportion of CD8 T cells
(Fig. 7, upper panel) but a slightly diminished proportion
of CD4 T cells. Staining with
H2-Kd/p60449–457 tetramers
shows that in excess of 90% of CD8 T cells from these mice stain with
the p60449–457 tetramers (Fig. 7, lower
panel), and that most of the Ag-specific cells express high levels
of CD62L.
To measure T cell proliferation in mice undergoing normal or
antibiotic attenuated bacterial infection, transgenic CD8 T cells
were stained with CFSE, a fluorescent dye that is incrementally diluted
with each cell division (25), and injected into naive
BALB/c recipient mice. Fig. 8
demonstrates that transgenic T cells transferred into mice were readily
detectable and maintained their high CFSE content 8 days later (Fig. 8A). Much larger p60449–457-specific
CD8 T cell populations were detected in recipients of transgenic cells
that were subsequently infected with L. monocytogenes (Fig. 8B). These expanded p60449–457 T cell
populations were CFSE negative, indicating that they had undergone
extensive in vivo division. We next transferred CFSE-labeled
TCR-transgenic T cells (expressing Thy1.2) into Thy1.1-congenic BALB/c
mice. One day later, mice were infected with L.
monocytogenes and then treated with ampicillin at 0, 12, or
24 h after bacterial inoculation. Splenocytes were harvested 5
days after infection, and CFSE staining of Thy1.2-positive,
p60449–457-specific T cells was measured by flow
cytometry. Similar to the findings in our investigation of the
endogenous LLO91–99-specific T cell response, we
found that the size of the p60449–457-specific T
cell response is dramatically influenced by the duration of live
bacterial infection during the first 24 h after inoculation of
L. monocytogenes. Mice that received ampicillin 24 h
after inoculation had maximal CD8 T cell responses to
p60449–457, whereas mice that received
ampicillin 12 h after inoculation had smaller T cell responses
(Fig. 8B). Importantly, although the responses in the
setting of early ampicillin therapy are smaller, the responding T cells
have all undergone extensive in vivo division since they have lost all
CFSE staining. Interestingly, 5 days after L. monocytogenes
infection, p60449–457-specific T cells fall into
one of two categories: those that have undergone extensive division
(>8–10 divisions) and those that have not divided at all. Thus, it
appears that cells that are activated undergo extensive division,
whereas others, which presumably received either no or a subthreshold
exposure to Ag, do not divide. It is noteworthy that undivided,
p60449–457-specific T cells persist in mice that
received no antibiotics even though they were exposed to extensive in
vivo bacterial infection. This result indicates that the extensive
bacterial growth that occurs between 24 h and 7 days after
inoculation does not result in progressive priming of naive T cells,
suggesting that in vivo T cell priming is temporally restricted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is possible that Ags produced by L. monocytogenes during the first 24 h of infection persist in a depot site for prolonged periods of time after infection. Furthermore, it is possible that secreted L. monocytogenes Ags are processed and presented in vivo long after the last viable bacterium has been killed by the host. Thus, it is not possible to state that Ag is absent in mice infected with low doses of L. monocytogenes or those treated with ampicillin at early time points following infection. In previous studies, however, we showed that the kinetics of the CD8 T cell response to peptides derived from three different L. monocytogenes Ags is similar (15). Therefore, if these Ags are driving the expansion and contraction of distinct CD8 T cell populations, it would indicate that these Ags are presented in vivo for similar periods of time. This seems highly unlikely, since Ags are synthesized by L. monocytogenes at vastly different rates, and their rate of degradation by mammalian cells is markedly different (26, 27). In the current study, we show that the size of T cell responses are similar in mice that are infected with nearly a 1000-fold range of L. monocytogenes doses. Additionally, we show that the size of Ag-specific T cell populations are similar in mice that have undergone normal or antibiotic-attenuated infection. It is highly unlikely that the duration of in vivo Ag presentation is similar in all of these circumstances. Thus, while it is possible that some Ags are present in vivo for much longer periods of time than the duration of viable infection, our results strongly argue that the duration and extent of T cell expansion are not simply determined by the presence or absence of cognate Ag.
At least two models can explain Ag-independent in vivo T cell expansion following bacterial infection. The first is that Ag-specific T cells are programmed during the first day of infection to undergo a given number of in vivo divisions. Once T cells are programmed, they are no longer dependent upon further Ag presentation. T cell proliferation following in vivo stimulation may be similar to the cellular programs or clocks that drive oligodendrocyte division and differentiation following exposure to growth factors and retinoic acid (28, 29). A second model to explain Ag-independent in vivo T cell proliferation is that an in vivo scaffold is generated during the first day of infection that promotes the expansion of effector and memory T cells. The scaffold would not depend upon continued in vivo infection to promote T cell expansion beyond the first day of infection. It is tempting to propose a structural scaffold that provides physical niches within lymphoid tissues for expanding Ag-specific T cell populations. An alternative idea, however, is that a soluble or cell-associated cytokine milieu that promotes lymphoid generation is generated during the first day of bacterial infection. TNF and related molecules have been implicated in the genesis of lymphoid tissues and likely play an important role in the generation of Ag-specific T cell populations (30, 31, 32). We suggest that the innate immune response to L. monocytogenes infection during the first day of infection creates a scaffold that determines the extent and duration of T cell expansion. The importance of innate immunity in determining T cell expansion kinetics is suggested by the finding that T cell responses to the same epitope presented in the context of infections by different pathogens results in T cell populations of markedly different magnitudes (33, 34).
The timing of in vivo T cell priming has been shown to dramatically influence the expansion of Ag-specific T lymphocytes (35). We extend this finding by showing that the size of the T cell response to two L. monocytogenes-derived epitopes is determined in the first day of infection and is tightly linked to the duration of live bacterial infection during the first 24 h after inoculation. Although we found that LLO91–99- and p60449–457-specific T cell responses are normal when ampicillin is administered 24 h after bacterial inoculation, administration of ampicillin 12 h after inoculation results in a significantly smaller LLO91–99-specific T cell response. This result is reminiscent of findings described by Sprent et al. (36) nearly 30 years ago when they demonstrated that systemic administration of Ag clears the thoracic duct lymph of Ag-specific T lymphocytes within 24–48 h. Thus, T lymphocyte circulation is sufficiently efficient that all Ag-reactive T cells come into contact with cognate Ag in a period as brief as 24 h. Our studies of LLO91–99- and p60449–457-specific responses also indicate that the maximal number of Ag-specific T cells can become activated with 24 h of infection. However, our studies transferring p60449–457-specific cells demonstrate that many residual Ag-specific T cells do not divide upon transfer. This result, while showing that the magnitude of a T cell response can be enhanced by increasing the precursor frequency of naive Ag- specific T cells, also suggests that the capacity to prime Ag-specific T cells is saturable. Saturation of the in vivo priming process has been previously suggested by the studies of Butz and Bevan (37) of lymphocytic choriomeningitis virus-specific T cell priming.
The finding that T cell expansion in response to L. monocytogenes infection is independent of prolonged in vivo Ag presentation is interesting in the context of recent studies of naive and memory T cell homeostasis. Naive T cells require MHC molecules for in vivo proliferation (38, 39, 40, 41) while memory T cells appear to undergo slow in vivo division without the need for any MHC-induced, TCR-mediated stimuli (42, 43). We do not know whether the expansion of Ag-specific T cells requires the presence of MHC class I molecules. It is possible that, upon activation, L. monocytogenes-specific T cells acquire proliferative characteristics that have been attributed to memory T cells, i.e., independence from TCR-mediated stimuli. Further studies will be required to answer this question.
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Acknowledgments |
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Footnotes |
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2 R.M. and S.V. contributed equally to the work described in this manuscript.
3 Current address: Henry M. Jackson Foundation for the Advancement of Military Medicine, 1600 East Gude Drive, Rockville, MD 20850.
4 Address correspondence and reprint requests to Dr. Eric G. Pamer, Infectious Diseases Service, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.
5 Abbreviation used in this paper: LLO, listeriolysin O.
Received for publication August 24, 2000. Accepted for publication September 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and 
T cells in immunity against an intracellular bacterial pathogen. Nature 365:53.[Medline]
and ß revealed in lymphotoxin ß deficient mice. Immunity 6:491.[Medline]
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 D. A. Blair and L. Lefrancois Increased competition for antigen during priming negatively impacts the |
) and the second group received
drinking water supplemented with 2 mg/ml ampicillin, beginning 24
h after bacterial inoculation (•). Spleens were harvested from three
mice per group at the indicated time points, homogenized, and the
number of viable L. monocytogenes was determined by
plating on brain-heart infusion plates. The mean number of bacteria per
spleen at the indicated time points was determined and plotted.
B, BALB/c mice were infected with L.
monocytogenes and either left untreated or treated with
ampicillin 24 h after infection. Seventy-two hours after
infection, spleens were harvested and the amount of IFN-
) or
treated with ampicillin 24 h after L. monocytogenes
inoculation (•). Spleens were harvested from three mice per group at
3, 7, 9, and 14 days after infection, stained, and analyzed flow
cytometrically as described in B, and the mean number of
LLO91–99-specific cells per spleen for each time point is
plotted. B, BALB/c mice were infected with L.
monocytogenes and either treated with ampicillin 24 h
after infection or left untreated. CD8 T cells were enriched from
splenocytes 7 days after infection and assayed for cytolytic activity
against 51Cr-labeled P815 target cells. The percent
specific lysis in the presence of LLO91–99 (
) or in the
absence (
