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CUTTING EDGE |
Infectious Diseases Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
| Abstract |
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| Introduction |
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8 days
(4, 5). A remarkable feature of this expansion is that CD8
T cells with different peptide specificities undergo synchronous in
vivo expansion, even though the peptides they respond to are present in
different amounts and form complexes with MHC class I molecules that
differ in stability (6, 7). Synchronous T cell expansion
and contraction in the face of disparate Ag quantity and stability
suggests that the duration of T cell expansion is not governed by the
presence or absence of Ag. More recent studies have also supported the notion that the extent of in vivo Ag presentation does not determine the duration of T cell expansion. Administration of bacteriocidal antibiotics that limit in vivo L. monocytogenes growth to 24 h following infection did not shorten the duration of in vivo T cell proliferation (8). Remarkably, the kinetics of CD8 T cell proliferation were unaltered whether in vivo infection with live bacteria progressed for only 1 or 7 days. Because the amount of Ag and the severity of inflammation are vastly different in these two circumstances, this study further supports the hypothesis that cell proliferation does not cease because Ag is depleted.
To explain T cell expansion during L. monocytogenes infection, we have proposed that T lymphocytes are programmed to undergo multiple rounds of division in an Ag-independent fashion following in vivo priming. To test this hypothesis, in the current report we stimulated Ag-specific T lymphocytes in vitro for short periods of time, removed them from Ag, and then characterized their proliferation. We find that transient in vitro stimulation for periods as short as 2.5 h is sufficient to program CD8 T lymphocytes to undergo up to eight cycles of division. These experiments unequivocally demonstrate that Ag is not required for T lymphocyte populations to expand and that generation of Ag-specific T cell populations can occur even when Ag presentation is transient. These findings have important implications for the development of protective immunity following infection and vaccination.
| Materials and Methods |
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BALB/cJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). WP11.12-transgenic mice expressing the p60449457/Kd-specific TCR were generated as previously described (8). RAW264.7 macrophage-like cells and P815 mastocytoma cells were obtained from American Type Culture Collection (Manassas, VA). All cell cultures were grown at 37°C, 5% CO2, in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with L-glutamine, HEPES, 2-ME, antibiotics (penicillin/streptomycin), and 10% FCS. L. monocytogenes strain 10403S was provided by Daniel Portnoy (University of California, Berkeley, CA).
Cell sorting and CFSE labeling
CD8 T cells were enriched to
90% purity from the spleen by
MACS (Miltenyi-Biotec, Auburn, CA) using anti-CD8a-conjugated
microbeads. Whole splenocytes or purified CD8 T cells were washed with
PBS and resuspended at 5 x 107/ml in PBS
containing 1 µM CFSE (Molecular Probes, Eugene, OR). The cell
suspension was incubated at 37°C for 10 min and immediately washed
with cold RPMI 1640/10% FCS before plating.
In vitro T cell stimulation
In peptide stimulation experiments, purified, CFSE-labeled WP11.12 CD8 T cells were plated at 2 x 106/well in 24-well plates containing 1 x 106 RAW264.7 cells previously pulsed or nonpulsed for 1 h with 10-6 M p60449457 peptide. After 24 h, CD8 T cells were recovered by MACS sorting and transferred to 96-well plates at 2 x 105/well along with 2 x 105 RAW cells pulsed or nonpulsed with peptide. In Ab stimulation experiments, purified, CFSE-labeled CD8 T cells were plated at 15 x 105/well on 96-well plates containing various concentrations of immobilized anti-CD3 and anti-CD28 mAbs. At the times indicated in the figure legends, T cells were transferred into new wells with or without Abs.
In splenocyte culture experiments, spleens were taken from BALB/c mice infected 3 days previously with 2000 L. monocytogenes. Dissociated spleen cells were enriched for CD8 T cells in some experiments, labeled with CFSE, and plated at 106/well in 24-well plates in RPMI 1640 medium with antibiotics. Proliferation was assessed daily by analysis of CFSE dilution by CD8 T cells over the course of a week.
Cytokines
Recombinant murine IL-7 and human IL-15 were obtained from R&D Systems (Minneapolis, MN) and were used at a final concentration of 12 ng/ml. Murine IL-2 was used at a concentration of 5 U/ml.
Abs, tetramers, and flow cytometry
The following mAbs directed to mouse cell surface Ags were
purchased from BD PharMingen (San Diego, CA): anti-CD8a-PerCP
(53-6.7), anti-CD69-PE (H1.2F3), anti-TCR
-APC (H57-597),
anti-CD3
(145-2C11), and anti-CD28 (37.51). PE-conjugated
streptavidin tetramers of H-2Kd class I MHC
complexed with listeriolysin O
(LLO)91993
peptide for detecting epitope-specific T cell populations were
generated as previously described (6). For four-color
analyses,
5 x 105 cells were incubated
on ice for 1 h with saturating concentrations of tetramers and the
various mAbs. Labeled cells were washed with PBS containing 1% FCS and
0.05% sodium azide and analyzed on a FACSCalibur flow cytometer
(Becton Dickinson, Mountain View, CA) using CellQuest software.
| Results |
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| Discussion |
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In this study, we show that transient antigenic stimulation of naive CD8 T lymphocytes induces their proliferation for up to 6 days following removal from antigenic stimulation. Although T cell proliferation is not dependent upon Ag, exogenous cytokines, particularly IL-2, are critical for continued T cell division. The size of the responding T cell population is positively influenced by the addition of either IL-7 or IL-15, two cytokines that have been implicated in CD8 T cell homeostasis (11, 12, 13, 14). Our findings suggest that these two cytokines may play a role in supporting the expansion of Ag-specific T cell populations during immune responses to infection.
Our results differ somewhat from those of a recent study suggesting that sustained TCR signaling of at least 6 h in the presence of costimulation is required for naive T cell proliferation, a process that is not significantly augmented by exogenous IL-2 (21). However, that report investigated CD4 T cell responses to cognate peptide-MHC complexes; it may be that stimulation with anti-CD3 mAbs in our study gives a stronger TCR signal that decreases the duration required for commitment to cell division. Also, we have not formally excluded the possibility that a longer duration of signaling may be required for proliferation under conditions of suboptimal IL-2 availability. In this regard, we have preliminary data demonstrating that CD8 T cells activated in vitro for 12 h can proliferate at least four times in vivo in naive mice in the absence of added exogenous IL-2. We are currently investigating the requirements for in vivo Ag-independent CD8 T cell expansion following in vivo priming.
The ability of CD8 T lymphocytes to undergo prolonged division in the absence of Ag provides an explanation for the finding that the kinetics of Listeria-specific CD8 T cell expansion are not influenced by the severity or duration of infection (8). Moreover, it is consistent with observations of synchronous T cell expansion to Ags that are present in vastly different amounts and that differ in their stability (6). Based on our findings, it is likely that infection with L. monocytogenes primes CD8 T cells during the first 2448 h and that subsequent expansion occurs regardless of whether or not Ag is further presented. We propose that T cell expansion is not driven by the continued presence of Ag, but rather occurs without any requirement for further signaling through the Ag receptor after the initial stimulation.
What factors are responsible for driving Ag-independent T cell
expansion following TCR activation? Cytokines clearly play an important
role in allowing survival and proliferation of activated T cells, and
it has been shown that cytokine-mediated signals can control lymphocyte
proliferation by regulating the expression of cell cycle proteins that
control entry into the S phase of the cell cycle (22). We
have shown that provision of at least IL-2 is necessary in our in vitro
system, which is consistent with other studies demonstrating its
importance in T cell proliferation (23, 24, 25). However,
other cytokines such as IL-7 and/or IL-15, which have been recently
implicated in regulating naive and memory T cell homeostasis
(11, 12, 13, 14), may play important roles for in vivo T cell
expansion following antigenic stimulation. Indeed, one recent report
has demonstrated that IL-15 may be critical for initiating T cell
division in vivo, whereas IL-2 may limit the magnitude of expansion of
cycling cells (26). Inflammatory cytokine and chemokine
secretion has been demonstrated in L. monocytogenes-infected
tissues and the local cytokine milieu likely provides the exogenous
stimuli that promote continued T cell division for 78 days following
initial infection. In support of this, we have recently shown that
increased expansion of activated T cells can be achieved by
inflammation induced by a second bacterial infection in the absence of
specific Ag (27). Whether IL-7 or IL-15 plays a role in
promoting in vivo proliferation of L. monocytogenes-specific
T lymphocytes during infection remains unknown and is currently being
investigated. Other factors such IFN-
, perforin, and costimulatory
signals through CD28 have also been shown to influence T cell expansion
(28, 29, 30) and may play roles in Ag-independent T cell
proliferation in vivo.
| Acknowledgments |
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| Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric G. Pamer, Infectious Disease Service, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. ![]()
3 Abbreviation used in this paper: LLO, listeriolysin O. ![]()
Received for publication March 20, 2001. Accepted for publication March 22, 2001.
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A. Ploss, I. Leiner, and E. G. Pamer Distinct Reg |