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Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, Minneapolis, MN 55455
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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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+ DC expressing the class I/peptide Ag complex via cross-presentation (6) can provide all three signals when activated through Toll-like receptors (TLR) to express high levels of CD80/CD86 and produce the signal 3 cytokine. In the absence of signal 3, recognition of Ag leads to the induction of tolerance even when levels of costimulatory ligands are high (2, 7, 8, 9). Stimulation by Ag in vivo in the absence of signal 3 results in multiple rounds of cell division, as indicated by CFSE dye dilution, but cell numbers do not increase, i.e., clonal expansion is impaired, suggesting that a major contribution of the third signal is to promote survival of the dividing cells (8). In addition, the cells do not develop cytolytic effector function when stimulated in the absence of a third signal. Several recent reports have provided evidence that a relatively brief exposure of naive CD8 T cells to Ag and costimulation initiates a program that drives the cells through multiple rounds of division and leads to development of effector function and a memory population. Using a system that used adherent cells transfected with Ag and B7-1 as APC, van Stipdonk et al. (10) showed that exposing naive CD8 T cells to the adherent APC for as little as 2 h and then placing the T cells in new cultures in the absence of Ag was sufficient to drive multiple rounds of division over the next several days. Similarly, studies by Pamer and colleagues (11, 12) and Ahmed and colleague (13) provided evidence that exposure to Ag for 24 h in vivo was sufficient to drive CD8 T cells through multiple rounds of division over the next few days. A review of this work by Bevan and Fink (14) termed this the "autopilot" response, i.e., a brief exposure to Ag activates the autopilot so that the cells undergo multiple divisions and differentiate in the absence of further stimulation. However, they pointed out that additional experiments would be needed to determine whether the size of the resulting effector and memory populations might be influenced by inflammatory cytokines or other signals. More recent studies have suggested, in fact, that a more prolonged interaction with Ag and costimulatory ligand may be necessary to achieve optimal clonal expansion and development of effector function (15, 16). None of these studies have explicitly accounted for the effects of signal 3 cytokines on the responses being measured.
We have conducted a series of experiments to determine when signal 3 (provided by IL-12) is required during the naive CD8 T cell response, and how this relates to the timing requirement for Ag exposure. The results demonstrate that brief exposure to Ag and costimulation, in the presence or absence of a signal 3 cytokine, does stimulate cell division but leads to little or no increase in cell numbers and the cells fail to develop effector functions. Optimal clonal expansion and acquisition of function require concerted signaling through the TCR, CD28, and IL-12R for at least 24–40 h. Thus, tolerance may occur as a result of stimulation by Ag and costimulatory ligand in the absence of signal 3 (8), or as a result of a brief exposure to all three signals. Furthermore, the abortive response that occurs upon brief exposure to Ag and costimulation cannot be "rescued" by subsequent signaling via a signal 3 cytokine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OT-I mice having a transgenic TCR specific for H-2Kb and OVA 257–264 were a gift from Dr. F. Carbone (University of Melbourne, Melbourne, Australia). OT-I mice were also crossed with Thy1-congenic B6.PL-Thy1a/Cy (Thy1.1) mice (The Jackson Laboratory, Bar Harbor, ME) and bred to homozygosity. The OT-I and OT-I/PL breeding colonies were maintained under specific pathogen-free conditions at the University of Minnesota. Experiments were performed in compliance with relevant laws and institutional guidelines and with the approval of the Institutional Animal Care and Use Committee of the University of Minnesota (Minneapolis, MN). C57BL/6NCr mice were purchased from the National Cancer Institute (Bethesda, MD). E.G7 tumor cells (EL-4 thymoma transfected with OVA) were used as targets in in vitro cytolysis assays and EL-4 cells were used as controls for specificity. All directly conjugated fluorescent Abs were purchased from BD PharMingen (San Diego, CA) or eBioscience (San Diego, CA). The medium used for all cell cultures was RPMI 1640 medium supplemented with 10% FCS, 4 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin and streptomycin, 10 mM HEPES, and 5 µM 2-ME (RP-10).
Naive T cell purification
Inguinal, axillary, brachial, cervical, and mesenteric lymph nodes (LNs) were harvested from OT-I or OT-I/PL mice, pooled, disrupted to obtain a single cell suspension, and were enriched for CD8+CD44low cells by negative selection using MACS magnetic cell sorting (Miltenyi Biotec, Auburn, CA). Briefly, cells were coated with FITC-labeled Abs specific for CD4, B220, I-Ab, CD11c, and CD44. Anti-FITC magnetic MicroBeads were added to the cells which were then passed over separation columns attached to the MACS magnet. The cells that did not bind to the column were collected and were >95% CD8+ and <0.5% CD44high. In some experiments, OT-I cells were labeled with CFSE before culture. OT-I cells were resuspended to 20 x 106/ml in HBSS and warmed in a 37°C water bath for 10 min before adding an equal volume of warmed 6 µM CFSE in HBSS and incubating for an additional 5 min at 37°C. Cells were washed three times with RP-10 before adding to microtiter wells.
Ag and B7-1 immobilization on microtiter wells
DimerX H-2Kb:Ig fusion protein (BD PharMingen) was diluted to 2 µg/ml in PBS and recombinant B7-1/Fc chimeric protein (R&D Systems, Minneapolis, MN) was diluted to 0.4 µg/ml in PBS. Wells of flat-bottom microtiter plates received 50 µl of each reagent and were incubated for 1.5 h at room temperature. Wells were washed twice with PBS, then 100 µl of RP-10 was added to each well and the plates were incubated overnight at 4°C. The next day, RP-10 was aspirated from the wells and peptide was loaded onto the H-2Kb portion of the fusion protein by adding 100 µl/well of OVA257–264 peptide diluted to 0.02 µM in RP-10. Wells were incubated for 2 h at 37°C, and then washed three times with RP-10 to remove unbound peptide. These wells are referred to as Ag/B7-coated wells.
In vitro proliferation and cytotoxicity assays
A total of 5 x 104 purified CD8+ T cells were placed in Ag/B7-coated flat-bottom microtiter wells in 200 µl of RP-10. At the times indicated, some cultures were supplemented with murine rIL-12 at 2 U/ml (Genetics Institute, Cambridge, MA) and/or neutralizing sheep antiserum to IL-12 (Genetics Institute) at 3.5 µg/ml. Where indicated, cells were resuspended at the indicated times after the initiation of culture and transferred to uncoated wells or to wells that had been coated with Ag/B7. Cell division after 3 days, as indicated by dilution of CFSE fluorescence, was monitored using a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Diego, CA). Viable cell recovery after 3 days of culture was used as the measure of clonal expansion (cell division + survival) and is reported as the number of live cells per milliliter of the original culture (starting concentration was 2.5 x 105 cells/ml). Cytolytic activity was determined in a standard 51Cr release assay using E.G7 cells as targets, with EL4 cells included as a control for specificity. Effectors and targets were incubated together for 4 h; triplicate wells of each E:T ratio were assayed. Results are shown as percent-specific 51Cr release as a function of the E:T ratio. Alternatively, data are expressed as lytic units per 106 effectors, where one lytic unit is defined as the number of effector cells needed to cause 30% specific 51Cr release from target cells.
Adoptive transfer and immunization of OT-I/PL transgenic cells
Pooled LN from OT-I/PL mice were disrupted to yield single cell suspensions and washed with PBS. Before transfer, the cells were analyzed by flow cytometry to determine the percentage of CD8+ cells. Their CD25, CD69, and CD44 phenotypes were determined to confirm that the cells that were transferred were not activated. A total of 1.5 x 106 CD8+ cells in 0.3 ml of PBS were transferred via tail vein injection into age- and sex-matched naive 6- to 8-wk-old C57BL/6 recipients. Recipient mice were then rested for 24 h before immunization. For immunization, 10 µg/mouse of OVA257–264 peptide (SIINFEKL; ResGen, Carlsbad, CA) were dissolved in PBS and injected via tail vein in a volume of 0.2 ml. At the same time as peptide, or after 24 or 48 h, some animals also received 1 µg of recombinant murine IL-12 (Genetics Institute) via tail vein in a volume of 0.2 ml. Mice were sacrificed for analysis 3 days after peptide immunization. Numbers of OT-I/PL cells were determined by flow cytometry analysis (see below) and cytolytic function was assessed by a standard 51Cr release assay performed as described above, except that effector and target cells were incubated together for 6 h.
Adoptive transfer of in vitro-activated OT-I/PL transgenic cells
CD8+CD44low OT-1/PL cells were purified from pooled LN cells as described above and labeled with CFSE. Cells were cultured in empty wells without IL-12 or in Ag/B7-coated microtiter wells and supplemented with rIL-12 at 2 U/ml. After 16 h, cells were removed from the wells, washed, and counted. CFSE fluorescence of the OT-I/PL cells was monitored to confirm that cells had not yet divided. A total of 1 x 106 cultured OT-I/PL cells in 0.3 ml of PBS were transferred via tail vein injection into age- and sex-matched naive 6- to 8-wk-old C57BL/6 recipients. Some mice that received OT-I/PL cells that had been exposed to Ag/B7 and IL-12 in vitro received, in the same injection, Ag in the form of 10 µg/mouse of OVA257–264 peptide along with 1 µg/mouse of rIL-12. Mice were sacrificed for analysis 3 days after in vivo transfer.
Flow cytometric analysis of transferred cells
Mice were sacrificed at the times indicated. Spleen cells and LN cells (pooled from axillary, brachial, cervical, inguinal, and mesenteric nodes) were counted by trypan blue dye exclusion to determine total viable cell counts, and were stained with Abs to CD8 and Thy 1.1 to label the transferred OT-I/PL cells. Stained cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences) to determine the percent of OT-I/PL cells in the transferred mice. The total number of OT-1/PL cells was determined by multiplying the percent of OT-I/PL cells by the number of cells recovered. In some experiments, the extent of cell division was assessed by determining the dilution of CFSE fluorescence in CD8+Thy1.1+ cells.
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Results |
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To examine the timing requirements for stimulation of naive CD8 T cells, highly purified naive OT-I TCR transgenic T cells (>95% CD8+, <0.5% CD44high) specific for H-2Kb/OVA257–264 (17) werestimulated with Ag and B7-1 ligand immobilized in flat-bottom microtiter wells at densities determined to stimulate optimal responses by OT-I cells in the presence of IL-12 (data not shown). Under the conditions used here, clonal expansion and cytolytic function are maximal by 64 h. This approach eliminates potential contributions from other cell types or ligands, and allows control of signal 3 by addition of IL-12 to the cultures. Furthermore, the duration of exposure to signal 3 can be controlled by delaying addition of IL-12 or by removing it by addition of neutralizing anti-IL-12 Ab. The use of plates bearing Ag and B7-1 ligand insures that effects of IL-12 result from its direct action on the CD8 T cells rather than its influence on levels of Ag or costimulatory molecules on APC.
When naive OT-I cells are stimulated for 64 h with Ag and B7-1 in the absence of IL-12, the cells fail to significantly increase in number or develop cytolytic activity, but respond strongly if IL-12 is added at the initiation of the cultures (i.e., at 0 h; Fig. 1, a and b). Delaying the addition of IL-12 to the cultures until 6 h after exposure to H-2Kb/Ag/B7 does not reduce the extent of peak clonal expansion at 64 h, but expansion begins to decline if IL-12 is not added until 18 h after exposure to Ag and costimulation (Fig. 1a). In contrast, development of lytic activity at 64 h was only weakly reduced when IL-12 addition was delayed until 18 h, and substantial lytic activity still developed when IL-12 addition was delayed until 40 h (Fig. 1b). Thus, strong responses could be obtained when IL-12 did not become available until somewhat later than Ag/B7. To determine how long the IL-12 needed to be present to support clonal expansion, neutralizing anti-IL-12 Ab was added to cultures at varying times after stimulation with Ag/B7 and IL-12. Adding anti-IL-12 Ab immediately almost completely eliminated increases in cell number, and substantial inhibition still occurred when the Ab was added at 6 or 18 h (Fig. 1a).
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Delaying addition of IL-12 until 40 h still resulted in substantial development of lytic function (Fig. 1d, solid line), but this declined rapidly if addition was delayed further. Conversely, neutralizing the IL-12 as late as 55 h still substantially reduced the lytic activity that developed (Fig. 1d, dotted line). Thus, IL-12 must be present between 
40 and 60 h to support strong development of lytic function. Similar results to those shown in Fig. 1, c and d, were obtained in five additional experiments examining the effects of varying the time of exposure to IL-12. Thus, prolonged exposure to IL-12 is required for both clonal expansion and development of lytic activity, but the "window" during which IL-12 must be present is later for development of lytic effector function. As a result, IL-12 must be present for most of the 3-day period needed for an optimal CTL response to be generated.
Although IL-12 is required for naive cells to increase in number, it is not required for the cells to be stimulated to undergo multiple rounds of cell division. When OT-I cells were labeled with CFSE and stimulated with Ag/B7, the extent of cell division, as indicated by dilution of the fluorescent dye, was very similar in the presence or absence of IL-12, with IL-12 causing at most one additional round of division (Fig. 1e). Thus, the need for prolonged exposure to IL-12 to support clonal expansion appears to result from it being required to promote increased cell survival rather than to increase the extent of cell division.
In vivo responses of CD8 T cells to challenge with peptide Ag normally require delivery of the Ag along with an adjuvant, but coadministration of IL-12 can bypass this requirement for adjuvant by directly providing signal 3 to the T cells (2, 3). To determine whether the timing requirements for IL-12 exposure defined above for in vitro responses were similar for in vivo responses, we examined the responses of adoptively transferred OT-I T cells upon challenge of the recipient mice with peptide and IL-12 administered at varying times. As previously seen, peptide alone stimulated only a small expansion in cell number (Fig. 2a) and the cells did not develop cytolytic function (Fig. 2b). Administration of IL-12 at either 0 or 24 h stimulated comparably strong clonal expansion to the peptide, while expansion was substantially less when IL-12 administration was delayed until 48 h (Fig. 2a). Some cytolytic activity developed in response to IL-12 administered at either 0 or 48 h, but was greatest when IL-12 administration was at 24 h (Fig. 2b). Although the time of exposure to IL-12 cannot be precisely controlled in vivo, these results are consistent with the in vitro results indicating that effective IL-12 signaling can begin after the initial exposure to Ag, and that the window for IL-12 signaling for optimal clonal expansion is earlier than for optimal development of cytolytic function.
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Recent studies have suggested that CD8 T cells may only need a brief exposure to Ag, as short as a few hours (10, 11, 12, 13), to be "programmed" to respond. Given the requirement for prolonged exposure to IL-12 for optimal responses, we re-examined how the duration of Ag exposure affected clonal expansion and cytolytic function. For these experiments, naive OT-I T cells were added to microtiter wells bearing H-2Kb/OVA257–264 Ag and B7.1 ligand and cultured for varying periods of time. The cells were then removed from the wells and placed in new wells that were either uncoated or had immobilized Ag and B7-1 ligand. At the end of 64 h, cell numbers were determined and cytolytic activity was measured. IL-12 was present throughout the 64 h of culture.
Continuous exposure to Ag and costimulation resulted in a large increase in cell number and development of cytolytic effector function (Fig. 3, a and b, condition 1). Exposure to Ag and costimulation for 6 h followed by transfer to uncoated wells, however, resulted in almost no clonal expansion or effector function, despite the continued presence of IL-12 (Fig. 3, a and b, condition 2). In contrast, when cells were transferred at 6 h to new wells having immobilized Ag/B7, the responses were essentially the same as for cells continuously exposed to the stimulus. Thus, transfer per se does not disrupt the responses. Although the cells did not expand in number or become cytolytic after 6 h exposure to Ag/B7 they did up-regulate surface expression of CD69 (Fig. 3c). Thus, failure to respond did not result from a failure to interact with the Ag during the brief exposure. When cells were allowed to interact with Ag/B7 for 16 h and then transferred to uncoated wells, responses increased substantially but were still significantly reduced in comparison to that of cells continuously exposed to Ag, or to cells transferred at 16 h to new wells bearing Ag/B7 (Fig. 3, a and b). All of the cells up-regulated CD69 surface expression after 16 h exposure to Ag, and the expression levels were somewhat higher than for 6 h exposure (Fig. 3c).
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24 h exposure to Ag/B7, while cytolytic activity requires 40 h to reach a maximum. Thus, as for IL-12 effects, development of optimal cytolytic activity requires somewhat longer exposure to Ag than does optimal clonal expansion. Experiments have also shown that after an initial 6 h exposure to Ag/B7, Ag alone is sufficient to provide the continuing stimulus needed to obtain maximal responses (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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24 h, while cytolytic function continued to increase with longer exposure times (Fig. 4). Signal 3 did not need to be present at the time of initial stimulation with Ag and B7-1 (Fig. 1a), and was not necessary to initiate cell division (Fig. 1e), but needed to be present from 20 to 40 h to support maximal clonal expansion, and from 40 to 60 h to support maximal acquisition of cytolytic function (Fig. 1, c and d). These results suggest a model in which brief interaction with Ag and B7-1 initiates a gene expression program that drives cells through multiple rounds of division without further stimuli being present, the previously described autopilot program (10, 11, 12, 13, 14). It appears that maintaining the expression of the genes needed for survival and development of function, however, requires prolonged signaling by a signal 3 cytokine. CD25, the -chain of the IL-2R, appears to be one such gene. In the absence of IL-12, CD25 expression is up-regulated on naive CD8 T cells in response to Ag/B7 x 24 h but then declines. In the presence of IL-12, expression continues to increase up to 48 h and is sustained at longer times (18). Furthermore, the results described here show that the signal 3 cytokine cannot act alone to maintain expression of the genes needed for survival and function, and instead concerted signaling stimulated by Ag, B7-1, and signal 3 cytokine is necessary. This model suggests that generating strong in vivo responses would require that the responding cells remain for a prolonged period of time at the site where Ag is being presented along with costimulatory ligands and where the signal 3 cytokine is being produced. This would normally occur in draining LNs where activated DC present Ag in the context of high levels of B7 ligands, and produce IL-12 in response to TLR ligation (4, 5). Early work by Sprent (19) showed that T cells are, in fact, sequestered for several days in draining LNs following Ag administration, and only then exit into the blood to traffic to peripheral sites. This is seen more directly in experiments using adoptive transfer of TCR transgenic CD8 T cells. Upon challenge of the recipients with peptide Ag in adjuvant, the cells undergo clonal expansion in the draining LNs that peaks at day 3, and only begin to exit the nodes in significant numbers on day 3 when cell division is largely complete and the cells have acquired effector function (2). Similarly, localized infection with HSV-1 results in initial activation of Ag-specific cells in the draining LNs, with activated cells not being detected in the spleen until 2 days later (20). Thus, it appears that there is a mechanism that insures that cells remain for a sufficient time at the site of Ag presentation to undergo the prolonged signaling needed to initiate productive responses.
Although presenting a quite different picture than that suggested by the initial autopilot reports (10, 11, 12, 13, 14), our results are largely consistent with those findings. Thus, we find that brief Ag exposure initiates cell division (Fig. 5), as reported by van Stipdonk et al. (10) who also examined OT-I cells. They did not report cell yields or extent of clonal expansion for their experiments. However, they did report that cells developed lytic function by day 3 after the 6 h exposure to Ag while we do not see this (Figs. 3 and 4). One possible explanation for this discrepancy is the fact that they used a JAM test (21) to measure cytotoxic activity, rather than a 51Cr release assay. This assay measures DNA fragmentation, and may detect Fas-dependent cytotoxicity; we have not examined Fas-dependent killing in our experiments. The experiments done by Ahmed and colleague (13) and Pamer and colleagues (11, 12) used Ag delivered in vivo by Listeria monocytogenes bacteria, which would be expected to stimulate some inflammatory response and likely cause production of IL-12. They showed that naive CD8 T cells were recruited to respond during the first 24 h of Ag exposure. Our results would predict that some level of response would be obtained within this time, but be suboptimal (Fig. 4). In fact, in one of the studies from Pamer and colleagues (11), the extent of clonal expansion after 24 h exposure is substantially reduced in comparison to continuous exposure.
More recently, van Stipdonk et al. (16) have examined the fate of OT-I T cells that were stimulated for varying periods of time in vitro with a fibroblast cell line expressing Ag and B7-1 and then adoptively transferred into normal mice, and found that stimulation for 20 h resulted in much greater survival and development of effector function than did stimulation for 4 h. Thus, with this stimulus also, brief Ag and B7-1 exposure initiates cell division but prolonged exposure is required for clonal expansion and development of strong effector function. Again, the potential role of signal 3 cytokines was not examined, but the results are consistent with our finding that prolonged Ag signaling is necessary even when a signal 3 cytokine is continuously available (Figs. 4 and 6). Finally, Gett et al. (15) have recently reported studies of CD4 and CD8 T cell activation from which they conclude that the strength of signal determines the fitness of the responding cells, with fitness defined as the ability to survive and respond to IL-7 and IL-15. Prolonged duration of signaling, considered as one of the measures of the strength of signaling, was found to be critical for conferring fitness, with exposure for 40–60 h giving optimal responses. In one experiment, immature and LPS-matured DC were compared as stimulator cells. Even when mature DC were used, which express high levels of Ag and costimulatory ligands and are likely to be producing signal 3 cytokines, prolonged interaction was required for optimal responses. Thus, prolonged signaling appears to be important even under optimal conditions of presentation by activated DC.
The realization that full activation of naive CD8 T cells, and avoidance of tolerance, requires prolonged and concerted signaling by Ag, costimulation, and signal 3 cytokine has obvious implications for development of effective immunization strategies. Our results also have important implications for understanding the gene expression programs involved in activating a full response, with the clear distinction between activation of cell cycle entry and division, which requires brief signaling via Ag and costimulation but not signal 3 cytokine, and activation of the gene expression programs that promote survival (and thus clonal expansion) and effector function, which require prolonged concerted signaling via Ag, costimulation, and a signal 3 cytokine.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Mayo Mail Code 334, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail address: mesch001{at}tc.umn.edu
3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; LN, lymph node.
Received for publication June 20, 2003. Accepted for publication September 9, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN-
by mouse dendritic cell subsets. J. Immunol. 166:5448.+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189:587.This article has been cited by other articles:
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D. L. Turner, L. S. Cauley, K. M. Khanna, and L. Lefrancois Persistent Antigen Presentation after Acute Vesicular Stomatitis Virus Infection J. Virol., February 15, 2007; 81(4): 2039 - 2046. [Abstract] [Full Text] [PDF]
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Q. Li, C. Eppolito, K. Odunsi, and P. A. Shrikant IL-12-Programmed Long-Term CD8+ T Cell Responses Require STAT4 J. Immunol., December 1, 2006; 177(11): 7618 - 7625. [Abstract] [Full Text] [PDF]
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S. Pejawar-Gaddy and M. A. Alexander-Miller Ligation of CD80 Is Critical for High-Level CD25 Expression on CD8+ T Lymphocytes J. Immunol., October 1, 2006; 177(7): 4495 - 4502. [Abstract] [Full Text] [PDF]
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M. Prlic, G. Hernandez-Hoyos, and M. J. Bevan Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response J. Exp. Med., September 4, 2006; 203(9): 2135 - 2143. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, D. C. Lins, C. M. Johnson, and M. F. Mescher Signal 3 Tolerant CD8 T Cells Degranulate in Response to Antigen but Lack Granzyme B to Mediate Cytolysis J. Immunol., October 1, 2005; 175(7): 4392 - 4399. [Abstract] [Full Text] [PDF]
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S. S. Pejawar, G. D. Parks, and M. A. Alexander-Miller Abortive versus Productive Viral Infection of Dendritic Cells with a Paramyxovirus Results in Differential Upregulation of Select Costimulatory Molecules J. Virol., June 15, 2005; 79(12): 7544 - 7557. [Abstract] [Full Text] [PDF]
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A. A. Filatenkov, E. L. Jacovetty, U. B. Fischer, J. M. Curtsinger, M. F. Mescher, and E. Ingulli CD4 T Cell-Dependent Conditioning of Dendritic Cells to Produce IL-12 Results in CD8-Mediated Graft Rejection and Avoidance of Tolerance J. Immunol., June 1, 2005; 174(11): 6909 - 6917. [Abstract] [Full Text] [PDF]
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R. Obst, H.-M. van Santen, D. Mathis, and C. Benoist Antigen persistence is required throughout the expansion phase of a CD4+ T cell response J. Exp. Med., May 16, 2005; 201(10): 1555 - 1565. [Abstract] [Full Text] [PDF]
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S. S. Kang and P. M. Allen Priming in the Presence of IL-10 Results in Direct Enhancement of CD8+ T Cell Primary Responses and Inhibition of Secondary Responses J. Immunol., May 1, 2005; 174(9): 5382 - 5389. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, J. O. Valenzuela, P. Agarwal, D. Lins, and M. F. Mescher Cutting Edge: Type I IFNs Provide a Third Signal to CD8 T Cells to Stimulate Clonal Expansion and Differentiation J. Immunol., April 15, 2005; 174(8): 4465 - 4469. [Abstract] [Full Text] [PDF]
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J. M. Roberts, J. Yang, and F. Ronchese IL-4 deficiency does not impair the ability of dendritic cells to initiate CD4+ and CD8+ T cell responses in vivo Int. Immunol., October 1, 2004; 16(10): 1451 - 1458. [Abstract] [Full Text] [PDF]
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A. T. Stock, S. N. Mueller, A. L. van Lint, W. R. Heath, and F. R. Carbone Cutting Edge: Prolonged Antigen Presentation after Herpes Simplex Virus-1 Skin Infection J. Immunol., August 15, 2004; 173(4): 2241 - 2244. [Abstract] [Full Text] [PDF]
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), are shown as a function of length of exposure to Ag and B7-1. For all time points, cells that were mixed and transferred to fresh wells coated with Ag and B7-1 showed the same response as cells that were not disturbed during the 64 h of culture (data not shown).
