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Deletion of Naive CD8 T Cells Requires Persistent Antigen and Is Not Programmed by an Initial Signal from the Tolerogenic APC ¹

William L. Redmond^*, Javier Hernandez and Linda A. Sherman^2,*

^* Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and Institut de Genetique Moleculaire de Montpellier, Montpellier, France

	Abstract

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Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naive CD8 T cells become activated when their TCR engages cognate peptide-MHC complexes on APCs. Depending upon several factors including the presence of costimulatory molecules and the maturation status of the APC, this encounter can result in either the development of an efficient CTL response or tolerance and deletion. Dendritic cells (DCs) ³ have been implicated as the key APCs and regulators of this process as they possess the unique ability to capture Ag from the parenchyma and present it to naive T cells circulating through secondary lymphoid tissues (1, 2). In addition, there is growing evidence that the activation status of DCs determines whether CD8 T cell activation results in a vigorous effector cell response or the induction of tolerance and deletion. Many different types of signals, including the presence of pathogens, proinflammatory cytokines, or the presence of activated CD4 T cells, lead to the up-regulation of costimulatory molecules, such as B7, by DCs (2, 3, 4, 5, 6). In the earliest stages of CD8 T cell activation, it is this up-regulation of B7 on DCs that costimulates T cells through CD28 and leads to enhanced T cell proliferation and survival. In contrast, CD8 T cell interactions with quiescent DCs that express basal levels of costimulatory molecules results in CD8 T cell deletion (7, 8, 9).

The model emerging from these reports is that DCs provide information that program CD8 T cell responses. Several recent studies provide support for this model by demonstrating that the strength of signal received by naive T cells during the initial encounter with APCs determines the extent of subsequent clonal expansion (10, 11, 12, 13, 14). Although this model accounts for differences in the magnitude of the initial CD8 T cell response, it is not known whether signals are also provided during an initial encounter with nonactivated DCs that program deletion.

To address whether the activation status of the DC differentially programs deletion vs survival of naive CD8 T cells, we compared the fate of CD8 T cells that were separated from the APC following activation under tolerogenic or stimulatory conditions in vivo. Our results support the paradigm that the activation status of the DC programs clonal expansion and the acquisition of CD8 T cell effector functions. However, regardless of whether CD8 T cells were stimulated under tolerogenic (quiescent DC) or immunogenic (activated DC) conditions, a portion of the T cells survive after removal from the presence of Ag. Therefore, although DCs are able to program the extent of proliferation, accumulation, and the acquisition of CD8 T cell effector functions, they do not determine the life or death of clonal progeny. These results highlight the importance of persistent Ag in promoting clonal deletion and anergy of self-reactive CD8 T cells.

	Materials and Methods

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Mice

BALB/c mice were purchased from the breeding colony of The Scripps Research Institute (La Jolla, CA). InsHA transgenic mice, homozygous for the hemagglutinin (HA) gene, and clone 4 TCR transgenic mice were generated and characterized as previously described (15, 16, 17). Clone 4 mice were also backcrossed with BALB/c mice for at least 10 generations and were then crossed with BALB/c Thy1.1 mice for two generations to obtain clone 4 TCR mice homozygous for Thy1.1. All mice were bred and maintained under specific pathogen-free conditions in The Scripps Research Institute’s animal facility. Experimental procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Purification and CFSE-labeling of naive clone 4 T cells

Single cell suspensions were prepared from the lymph nodes of clone 4 Thy1.1 TCR transgenic mice. Cell suspensions were depleted of CD4⁺, CD11b⁺, CD45R⁺, DX5⁺, and Ter-119⁺ cells using the MACS CD8⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Naive clone 4 Thy1.1 T cells were purified by negative selection per the manufacturer’s instructions and were CD44^low, CD62L^high, and CD69-negative as indicated by flow cytometry using FITC-conjugated Abs (BD PharMingen, San Diego, CA) (data not shown).

Purified clone 4 CD8 Thy1.1 T cells were washed once in cold HBSS and then resuspended at 5 x 10⁷ cells/ml in HBSS. A total of 2 µl of a 5 mM solution of CFSE (Molecular Probes, Eugene, OR) in DMSO (Sigma-Aldrich, St. Louis, MO) was added per milliliter of cells and incubated for 10 min at 37°C. Cells were then washed once with ice-cold HBSS. A total of 3 x 10⁶ CFSE-labeled clone 4 T cells were injected i.v. in 200 µl of HBSS.

Activation of clone 4 T cells

After adoptive transfer, naive clone 4 CD8 Thy1.1 T cells were activated under the following conditions. For viral priming, 500 hemagglutinating units (HAU) of influenza virus strain A/PR/8/34 H1N1, that was grown in the allantoic cavity of 10- to 11-day-old hen’s eggs, was injected i.p. into mice. HA peptide (⁵¹⁸IYSTVASSL⁵²⁶) was synthesized by the core facility of The Scripps Research Institute using an Applied Biosystems model 430A synthesizer (Foster City, CA). Purity was >85%, as determined by mass spectrometry and reversed-phase HPLC analysis on a Vydac C₁₈ column (Vydac, Hesperia, CA). Peptide was given by i.v. injection of 100 µg of K^d HA peptide in HBSS. For anti-CD40 mAb treatments, 150 µg of purified anti-CD40 mAb (FGK-45) in HBSS was injected i.p. into recipient mice. CD40 mAb was kindly provided by Dr. S. Schoenberger (La Jolla Institute for Allergy and Immunology, La Jolla, CA). Abs, peptide, and virus were injected on day 0.

Flow cytometry

Spleens, pancreatic and peripheral lymph nodes including inguinal, axillary, cervical, mandibular, and mesenteric were harvested and processed to obtain single cell suspensions. RBCs were lysed with an ammonium chloride lysing reagent, PharM Lyse, according to manufacturer’s instructions (BD Biosciences, San Diego, CA). Cells were rinsed three times with RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% heat-inactivated FCS (Gemini Bio-Products, Calabasas, CA) supplemented with 2 mM glutamine (Life Technologies) and 50 mg/ml gentamicin (Gemini Bio-Products). All mAbs and secondary reagents used for FACS analysis were purchased from BD PharMingen. Cells were incubated for 30 min on ice with anti-Thy1.2 FITC, anti-Thy1.1-PE, or anti-CD8-PerCP. After washing three times with HBSS containing 0.1% w/v BSA (Sigma-Aldrich) and 0.02% w/v sodium azide, cells were analyzed with a FACSCalibur and CellQuest software (BD Biosciences). To measure Ag-specific production of IFN-, LN cells or splenocytes were incubated in RPMI 1640 10% FCS with 1 µg/ml of the K^d HA peptide and 1 µl/ml of brefeldin A containing Golgi-Plug solution (BD PharMingen) for 6 h at 37°C. After washing, cells were stained to detect CD8 and Thy1.1 as described above. Cells were then permeabilized and stained to detect intracellular IFN- with IFN--APC mAb using the Cytofix/Cytoperm Plus kit (BD PharMingen) according to the manufacturer’s instructions.

In vivo cytolytic assay

A total of 3 x 10⁶ nonlabeled, purified clone 4 T cells were injected into recipient mice. Syngeneic spleen cells were labeled by incubation for 10 min at 37°C with either 5 µM CFSE in HBSS (CFSE^high cells) or 0.5 µM CFSE in HBSS (CFSE^low cells) and washed twice with HBSS. CFSE^high cells were pulsed with K^d HA peptide at 1 µg/ml for 1 h at 37°C. CFSE^low cells were not pulsed with peptide and served as an internal control. On day 2 or 4, a mixture of 3 x 10⁶ CFSE^high peptide-pulsed cells plus 3 x 10⁶ CFSE^low nonpulsed cells were injected i.v. into recipient mice. LN cells were harvested 10 h later and single cell suspensions were analyzed for detection and quantification of CFSE-labeled cells by flow cytometry.

DC isolation and analysis

Lymph nodes were harvested from InsHA or BALB/c mice treated as described above. Lymph nodes were mechanically disrupted and digested with collagenase-D (1 mg/ml) (Roche, Mannheim, Germany) for 60 min at 37°C in RPMI 1640 medium supplemented with 2% FCS. Following digestion, EDTA was added to the suspension to stop the collagenase activity. Lymph nodes were processed to obtain single cell suspensions and after washing, cells were stained with anti-CD11c-APC, anti-CD8-PerCP and anti-B7.1-PE (BD PharMingen) for 30 min on ice. Cells were washed two times and analyzed by flow cytometry.

	Results

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Activation and recovery of Ag-experienced clone 4 T cells

Our goal in this study was to test the hypothesis that during the initial encounter with Ag the activation status of the APC determines whether naive CD8 T cells undergo tolerance/deletion or survival. Our expectation was that activation under tolerogenic conditions, such as exposure to cross-presented self-Ag or high concentrations of soluble peptide Ag, would program cells for deletion. Conversely, we predicted that stimulation under immunogenic conditions, such as exposure to viral infection or Ag in the presence of anti-CD40 mAb, both of which result in increased expression of costimulatory molecules on APCs, would protect cells from deletion. To this end, we compared the fate of HA specific CD8 T cells derived from clone 4 TCR transgenic mice (15) after their stimulation in vivo under conditions known to promote either tolerance or immunity.

CD8 Thy1.1 T cells from clone 4 TCR mice (clone 4 T cells) were labeled with CFSE and adoptively transferred into Thy1.2 InsHA mice that express the viral HA on their pancreatic islet cells (17). Four days later, pancreatic lymph nodes were harvested and the proliferation profiles of donor clone 4 T cells were analyzed via flow cytometry. As demonstrated previously, cross-presentation of HA in the pancreatic lymph nodes of InsHA mice leads to the activation of clone 4 T cells, however, the cells do not accumulate, but rather, are tolerized and deleted (16, 18). In contrast, if at the time of cell transfer InsHA mice receive anti-CD40 mAb, a powerful activator of DCs (19, 20), then the clone 4 T cells proliferate more vigorously (Fig. 1A) and accumulate in greater numbers (19). Consistent with our previous studies, the presence of anti-CD40 results in the activation of the DCs that present HA in the pancreatic lymph nodes by up-regulating expression of B7 costimulatory molecules (Table I) which results in enhanced T cell proliferation and accumulation (19). In both cases, the amount of endogenous Ag that was cross-presented was insufficient to result in the activation of the majority of clone 4 T cells within the first few days in vivo (Fig. 1A).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Activation and recovery of clone 4 T cells. A, A total of 3 x 106 purified CFSE-labeled clone 4 CD8 Thy1.1 T cells were injected into Thy1.2 InsHA or BALB/c mice on day 0. At the time of transfer, clone 4 T cells were activated with endogenous Ag only (InsHA), 100 µg Kd HA peptide in HBSS, 150 µg anti-CD40 in HBSS, or 500 HAU of influenza virus. Four days later, pooled pancreatic (InsHA) or peripheral (peptide, virus) lymph nodes were harvested and the proliferation of donor clone 4 T cells was measured by flow cytometry. B, Activated clone 4 T cells (as described in Fig. A) were sorted by flow cytometry and 1 x 104 clone 4 T cells were adoptively transferred into nonirradiated BALB/c hosts. Only cells that had undergone a minimum of three divisions were collected. Four weeks after transfer, mice were immunized with influenza virus to induce expansion of the donor cells. Eight days after priming, spleens were harvested and clone 4 T cells were detected and quantitated by flow cytometry. The mean ± SD of four mice per group is indicated. C, clone 4 T cells from B were assessed for the production of IFN- following re-stimulation in vitro with Kd HA peptide for 6 h at 37°C. The mean ± SD of three to four mice per group is indicated. Data is representative of one of three independent experiments with similar results.

The adoptively transferred activated clone 4 T cells were rested in vivo for 4 wk, at which time recipient mice were immunized with influenza virus to induce sufficient expansion of the donor clone 4 T cells to allow their detection by flow cytometry. This was found to be necessary as the small numbers of transferred cells could not be detected without first undergoing expansion in response to viral priming (data not shown). Optimal expansion of the previously activated clone 4 T cells occurred eight days after priming and therefore, we harvested spleens and quantified activated donor clone 4 T cells at that time point. As expected, clone 4 T cells originally activated in InsHA mice in the presence of anti-CD40 mAb survived following adoptive transfer into Ag-free recipients (Fig. 1B). Surprisingly, clone 4 T cells activated by cross-presented self-Ag under tolerogenic conditions were also able to survive, although fewer cells were recovered (Fig. 1B). These results suggested that the cells initially activated by "tolerogenic" DCs were not all destined to undergo deletion.

As these results were unexpected, we wished to determine the generality of these findings by using different conditions of activation to initiate tolerance or immunity. Two activating conditions were selected in which high levels of exogenously provided Ag induce extensive proliferation of clone 4 T cells (9). clone 4 T cells were adoptively transferred into BALB/c mice and activated with 100 µg of soluble K^d HA peptide, a tolerizing condition, or by infection with influenza virus (Fig. 1A). As expected, viral priming, but not peptide treatment, led to the up-regulation of costimulatory molecules on the DCs (Table I) (23, 24). Activated clone 4 T cells that had undergone at least three divisions were collected and adoptively transferred into BALB/c recipients. Following transfer into Ag-free hosts, clone 4 T cells activated previously with soluble HA peptide or virus could be recovered (Fig. 1B). Thus, regardless of the activation status of the initial stimulatory APC, Ag-experienced clone 4 T cells could survive in Ag-free hosts. Importantly, the previously activated clone 4 T cells were functional as assessed by their ability to proliferate and produce IFN- in response to priming with virus (Fig. 1, B and C).

To confirm whether the initial activation conditions were truly tolerogenic or stimulatory, the effector status of activated clone 4 T cells was assessed by measuring both their ability to produce IFN- and in vivo cytolytic function. Consistent with our previous reports, activation of clone 4 T cells by endogenous cross-presented Ag or soluble HA peptide did not give rise to the production of IFN- or in vivo killing (Table I) (9). In contrast, anti-CD40 led to a slight increase and viral priming to a substantial increase in both IFN- production and cytolytic activity (Table I) (9).

Chronic Ag presentation is required for clone 4 CD8 T cell deletion

Thus far, our results suggested that the activation status of the APC was not sufficient to program clone 4 T cells for deletion. The main difference between the current study and previous studies in which we demonstrated tolerance and deletion of clone 4 T cells in InsHA mice was that in the current protocol, activated clone 4 cells were removed from the source of Ag (InsHA mice) and allowed to rest in Ag-free hosts. Therefore, we sought to test directly whether chronic antigenic stimulation was required to promote the deletion of clone 4 T cells.

Clone 4 T cells were activated as described (see Fig. 1A) and 10,000 activated clone 4 T cells were adoptively transferred into Ag-free BALB/c, InsHA (endogenous Ag only), or BALB/c mice receiving soluble HA peptide. Four weeks after the transfer, recipient mice were primed with the influenza virus and eight days later, the presence of donor clone 4 T cells was determined by flow cytometry. clone 4 T cells originally activated by endogenous Ag in the pancreatic lymph nodes of InsHA mice were readily detected following adoptive transfer into Ag-free mice (Fig. 2). In contrast, when transferred into InsHA mice (expressing endogenous Ag only) no clone 4 T cells were recovered following viral priming (Fig. 2). Similar results were obtained with clone 4 T cells activated in the pancreatic lymph nodes of InsHA mice in the presence of the anti-CD40 mAb. When placed into Ag-free recipients, but not mice expressing the endogenous HA Ag, previously activated clone 4 T cells could be recovered (Fig. 2). clone 4 T cells activated with soluble HA peptide were transferred into either Ag-free BALB/c mice or BALB/c mice that received 100 µg of HA peptide. Consistent with the results obtained with exposure to endogenous Ag, previously activated clone 4 T cells could only be recovered from Ag-free recipients and not from mice that received the HA peptide (Fig. 2). Thus, chronic exposure to Ag is required to promote the deletion of naive clone 4 T cells.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Chronic Ag presentation promotes clone 4 CD8 T cell deletion. Activated clone 4 CD8 Thy1.1 T cells (as described in Fig. 1A) were sorted by flow cytometry and 1 x 104 clone 4 T cells were adoptively transferred into nonirradiated recipients. Ag-experienced clone 4 CD8 T cells activated by endogenous HA in the pancreatic lymph nodes of InsHA mice (± anti-CD40 mAb treatment) were transferred into either Ag-free Thy1.2 BALB/c or InsHA mice. Ag-experienced clone 4 T cells activated by soluble Kd HA peptide in the lymph nodes of BALB/c mice were transferred into Ag-free BALB/c or BALB/c mice that received 100 µg Kd HA peptide on day 0 (at the time of cell transfer), day 7, and day 14. Four weeks after transfer, mice were immunized with influenza virus to induce expansion of the donor clone 4 T cells. Eight days after priming, spleens were harvested and clone 4 T cells were detected and quantitated by flow cytometry. Data is representative of one of two independent experiments with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have addressed the question of whether the activation status of the APC alters the ability of the T cell to survive after antigenic stimulation. Specifically, we wished to determine whether activation of a T cell by a quiescent APC was, in effect, a death sentence for the T cells. To this end, we collected clone 4 T cells that had been activated by either quiescent or activated APCs. Two different methods were used to stimulate HA-specific clone 4 T cells with quiescent APCs, cross-presentation in InsHA recipients or injection of HA peptide into BALB/c mice. Consistent with our previous findings, neither type of stimulation resulted in effector cell generation (Table I) (9). We also used two methods of activating T cells in vivo with activated APCs that had different consequences for the resultant acquisition of CD8 T cell effector function. As shown previously, the DCs that cross-present Ag in the pancreatic lymph nodes can be activated by anti-CD40 to up-regulate costimulatory molecules (Table I) and support accumulation of activated clone 4 T cells (19). Under these conditions, however, clone 4 T cells do not become potent effectors (9). In contrast, APCs activated by influenza virus promote both the accumulation of activated clone 4 T cells and development of effector function (9).

Although we observed major differences in the amount of clonal expansion and effector function among the various stimulatory conditions, in all cases a subset of the previously activated clone 4 T cells could survive after they were removed from the presence of Ag. Furthermore, when tested 1 mo later for their ability to clonally expand in response to viral challenge, Ag-experienced clone 4 T cells remained functional as assessed by their ability to proliferate and produce IFN- (Fig. 1, B and C). Therefore, the activation status of the DCs did not impact the subsequent ability of Ag-experienced clone 4 T cells to survive or respond to Ag.

Due to the small numbers of activated clone 4 T cells transferred into Ag-free hosts, it was not possible to quantitate the proportion of cells transferred at day 4 after initial activation that survived 1 mo posttransfer. However, based on the fact that most activated T cells undergo apoptosis, regardless of the type of stimulatory signal they receive (25), it is likely that only a minority of the cells survived. Although the numbers of cells recovered after viral infection varied from experiment to experiment (Fig. 1B), it did appear that fewer of the cells that underwent their initial activation in InsHA mice were eventually recovered when compared with the number recovered after activation in InsHA mice that were also given anti-CD40. This may be due to differential survival or to greater continued clonal expansion in the Ag-free host by the cells initially activated in the presence of anti-CD40. In comparing the numbers of cells recovered after initial activation with either virus or peptide (Fig. 1B), similar numbers of cells survived regardless of the activation status of the APC. Taken together, these experiments suggest that the activation status of the APC did not determine survival of the activated CD8 T cells.

Importantly, we were able to obtain complete tolerance and deletion of clone 4 T cells, but only upon transfer of previously activated clone 4 T cells into recipients that contained HA expressed either as a self-Ag, or through the injection of soluble HA peptide (Fig. 2). These results are consistent with the role of chronic Ag presentation in promoting CD8 T cell deletion as demonstrated previously using multiple injections with peptide to achieve tolerance of CD8 T cells specific for the lymphocytic choriomeningitis virus glycoprotein (26, 27). It will be of interest to determine the length of time required to promote deletion through TCR signaling. In our experiments, we can estimate that the time it takes for cells to have undergone at least three divisions (>48 h) is not sufficient (data not shown). Alternatively, it may be found that deletion requires repetitive as opposed to persistent encounter with Ag. Future studies will examine this issue.

It is of interest to speculate on whether persistent Ag would also lead to CD8 T cell deletion whether Ag were presented by an activated rather than quiescent APC. This is difficult to assess because activated APCs drive the development of CD8 T cell cytolytic activity, which, in turn, would lead to the clearance of Ag. Indeed, recent findings have shown that CD8 T cells that are activated in response to a pathogen such as Listeria monocytogenes or malaria quickly kill APCs (28, 29). Such removal of APCs may be an important part of the strategy by which the immune system prevents deletion of primed effector and memory cells.

Recent studies have provided evidence that the development of memory CD8 T cells requires the participation of CD4 T cell help (30, 31, 32). It is unlikely that such help was available in our experiments. As shown previously, InsHA mice are tolerant of HA in both their CD4 and CD8 compartments, which severely limits their ability to respond to HA (17). BALB/c mice primed with the K^d-restricted HA peptide would not have a source of Ag to stimulate HA specific CD4 cells. Although the BALB/c mice that received clone 4 T cells and influenza virus would be expected to have virus specific CD4 cells in their endogenous repertoire (33, 34), it is unlikely that significant numbers would have been available to provide help for the number of clone 4 T cells that were present in the influenza infected mice. Therefore, it is possible that the previously activated clone 4 T cells that persisted in the absence of Ag were not equivalent to true memory cells and despite their previous activation, may have functional properties similar to those of naive cells (35).

Several studies have suggested that the duration of the T cell-APC interaction is critical in promoting the acquisition of T cell "fitness" and providing T cells with the capability to respond to survival signals in vivo (13, 14). Those results suggest a model of T cell activation in which naive CD8 T cells require sustained TCR stimulation to differentiate into effector cells. The "fitness" model predicts that CD8 T cells receiving brief or weak antigenic stimulation do not survive in vivo. Our results indicate that although tolerogenic stimulation eventually does lead to deletion, this process requires the continuous presence of Ag beyond that which was required to dictate the extent of proliferation and gain of effector function. In the absence of Ag, previously activated CD8 T cells that had been exposed to Ag for sufficient time to undergo at least three divisions (>48 h) in vivo are not all deleted and many can survive for at least 1 mo and retain the ability to respond to a secondary challenge with Ag. Even cells that did not fully differentiate into effectors following the primary stimulation could later respond to viral priming.

In conclusion, these results indicate that the conditions under which naive CD8 T cells are initially activated do not program them for deletion after leaving the APC. Thus, stimulation of naive CD8 T cells under tolerogenic conditions with quiescent APCs does not commit their progeny to an irreversible death pathway. Rather, tolerance and deletion require the sustained presence of Ag. This stringent requirement for clonal deletion may help to maintain diversity in the CD8 T cell repertoire. If transient exposure to Ag programmed CD8 T cell deletion, potentially useful clones may be unnecessarily eliminated from the repertoire.

	Acknowledgments

 
We thank Dr. Stephen Schoenberger for providing the anti-CD40 mAb; Judith Biggs, Kristi Marquardt, and Rebecca Trenney for excellent technical assistance; The Scripps Research Institute Flow Cytometry core facility for assistance with cell sorting; and members of the Sherman Laboratory for helpful discussions.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants DK50824, DK57644, CA57855 (to L.A.S.), T32 AI07606, and an Achievement Rewards for College Scientists Foundation Fellowship (to W.L.R.).

² Address correspondence and reprint requests to Dr. Linda A. Sherman, Department of Immunology IMM-15, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: lsherman{at}scripps.edu

³ Abbreviations used in this paper: DC, dendritic cell; HA, hemagglutinin; HAU, hemagglutinating unit.

Received for publication July 22, 2003. Accepted for publication October 10, 2003.

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