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The Journal of Immunology, 2007, 179, 2870-2879
Copyright © 2007 by The American Association of Immunologists, Inc.
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Dynamic Control of Self-Specific CD8+ T Cell Responses via a Combination of Signals Mediated by Dendritic Cells1

Ben J. E. Raveney and David J. Morgan2

Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is acknowledged that T cell interactions with mature dendritic cells (DC) lead to immunity, whereas interactions with immature DC lead to tolerance induction. Using a transgenic murine system, we have examined how DC expressing self-peptides control naive, self-reactive CD8+ T cell responses in vitro and in vivo. We have shown, for the first time, that immature DC can also stimulate productive activation of naive self-specific CD8+ T cells, which results in extensive proliferation, the expression of a highly activated cell surface phenotype, and differentiation into autoimmune CTL. Conversely, mature DC can induce abortive activation of naive CD8+ T cells, which is characterized by low-level proliferation, the expression of a partially activated cell surface phenotype which does not result in autoimmune CTL. Critically, both CD8+ T cell responses are determined by a combination of signals mediated by the DC, and that altering any one of these signals dramatically shifts the balance between autoimmunity and self-tolerance induction. We hypothesize that DC maintain the steady state of self-tolerance among self-specific CD8+ T cells in an active and dynamic manner, licensing productive immune responses against self-tissues only when required.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To mount an appropriate immune response, yet maintain a steady state of self-tolerance, it is crucial that the immune system is able to distinguish between self- and foreign Ags. Classically, dendritic cells (DC)3 are involved in the initiation of immunity to pathogens (1, 2, 3). However, Ag presentation by DC does not always generate productive immunity. In vivo, it has been shown that DC are able to cross-present exogenous microbial Ags as well as self-Ags to naive CD8+ T cells (4, 5). Yet in the steady state, DC initiate CTL responses to foreign Ags and presentation of self-Ags by DC may result in tolerance induction (6, 7, 8, 9, 10, 11, 12).

Several suggestions have been made to explain these opposing T cell responses. Steinman et al. (13) have suggested that the maturation state of a DC is key in determining the outcome of T cell activation. Mature DC (mDC), which express high levels of costimulatory molecules such as CD80 and CD86, prime T cell responses, while immature DC (iDC), which express lower levels of costimulatory molecules, may induce tolerance (13). DC generated from ex vivo culture of bone marrow (BM) cells can be matured by treatment with bacterial LPS (mDC) (14). These mDC have been shown to activate naive T cells in vitro to form CTL (15), whereas, in the absence of maturation signals, ex vivo-generated DC were unable to initiate CTL responses (16). When OVA protein was targeted to murine DC by administering an OVA-conjugated anti-DEC-205 (17) mAb (OVA-anti-DEC-205), OVA-specific OT-1 CD8+ T cells were deleted from the peripheral repertoire without inducing effector CTL responses. However, when anti-CD40 mAbs, which initiate DC maturation (18), were also administered at the same time as OVA-anti-DEC-205, OT-1 effector CTL were generated (19). Other studies have indicated that the CD8{alpha}+ subset of DC is involved in inducing self-tolerance among naive CD8+ T cells in vivo (11, 12, 20). However, it is likely that in these experiments the tolerizing CD8{alpha}+ DC described were also immature. Further studies have also demonstrated that the tolerizing properties of DC are associated with their anatomical location. Clearly, presentation of self-Ags by DC in the thymus leads to the induction of self-tolerance (6, 7). However, a recent study has shown that DC expressing the tolerogenic protein indoleamine dioxygenase are only observed in splenic tissue (21). Thus, it seems likely that the outcome of naive T cell interactions with DC (22) are influenced by a combination of factors which include: the cell surface phenotype, state of maturation, and the anatomical location of the DC. Moreover, the prevalence of certain subsets of DC with a particular cell surface phenotype may indeed be related to the maturation state of the DC, and the maturation state of the DC may also be influenced by the microenvironment within which it resides. However, it is unclear whether the maturation state of the DC is the overriding factor governing the outcome of CD8+ T cell-DC interaction. Furthermore, if the level of activation falls below a certain threshold, then is tolerance the only possible outcome? Conversely, if activation occurs above this threshold, then is the only outcome productive activation and the formation of CTL? Alternatively, tolerance induction and the formation CTL may represent discrete clinical readouts among a broad range of different T cell responses based on their proliferation and cytokine secretion.

To address these important issues, we used CL4 CD8+ T cells which express a transgenic TCR which recognizes, with high avidity, the dominant H-2Kd-restricted epitope of the hemagglutinin (HA) glycoprotein from influenza virus A/PR/8 H1IN1 (PR8) (23). Previous studies in vivo have shown that the activation of naive CL4 CD8+ T cells can result in entirely opposing responses (24). When transferred into transgenic InsHA mice, which express HA as a neo-self-Ag solely on pancreatic islet β cells, CL4 CD8+ T cells are activated only in the pancreatic lymph nodes (PLN) and, although they proliferate, they do not differentiate into effector CTL (25). As a result, host InsHA mice do not develop autoimmune diabetes. Such activation is termed "abortive" and is thought to be responsible for the functional loss of HA-specific CD8+ T cells, which accompanies the induction of peripheral tolerance in InsHA mice (26). However, if InsHA recipients are immunized with PR8 at the time of CL4 CD8+ T cell transfer, then productive activation of CL4 CD8+ T cells occurs leading to the generation of HA-specific CTL and autoimmune diabetes (23).

We have observed that abortive activation results in the up-regulation of cell surface Ly6C expression among CD8+ T cells. Previous studies have shown that there may be association of Ly6C expression with memory and effector function by T cells (27); however, other studies have suggested that Ly6C is associated with decreased effector function (28). Ly6C may also have a role in tolerance induction, as evidenced by the fact that certain mouse strains that are prone to the autoimmune diseases type 1 diabetes mellitus or systemic lupus erythematosus (NOD and New Zealand Black (NZB), respectively), have a mutation in Ly6C (29). Furthermore, Ly6C expression has been observed among the plasmacytoid DC population (30) which is thought to induce peripheral T cell tolerance by generating regulatory T cells (31, 32). Our data now suggest that Ly6C expression may also be important hallmark of CD8+ T cells undergoing peripheral tolerance induction via abortive activation.

Significantly, the data presented in this report show for the first time that iDC, although capable of inducing abortive activation of naive CL4 CD8+ T cells both in vivo and in vitro, under certain conditions, are as effective as mDC in inducing productive activation to form CTL. Furthermore, we have shown that under other conditions, mDC activation of naive CD8+ T cells does not result in CTL induction. These different responses are governed by a combination of signals 1, 2, and 3 mediated by the DC via MHC/peptide, costimulatory molecules, and cytokines, respectively, and that altering the level of any one of these signals can result in a shift in the balance of the response between either abortive or productive activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c mice were purchased from the University of Bristol Animal Services Unit. BALB/c InsHA-transgenic mice and Thy1.1+ BALB/c CL4 TCR-transgenic mice were generated and characterized as previously (24, 33). All mice were bred and housed under specific pathogen-free conditions at the University of Bristol Animal Service Unit. Experimental procedures were conducted according to U.K. Home Office guidelines.

Generation of BM-derived DC

BM-derived iDC were generated in vitro from hemopoietic progenitor cells using a method adapted from Lutz et al. (14). BM cells were extracted by flushing the long bones of 6- to 8-wk-old BALB/c mice with HBSS (Invitrogen Life Technologies) plus 10% v/v FCS (Invitrogen Life Technologies). BM cells were disaggregated and resuspended at 4 x 105 cells/ml in complete RPMI (RPMI 1640 supplemented with 2 mM glutamine, 50 U/ml penicillin/streptomycin, 0.1 mM 2-ME, and 10% v/v FCS; all obtained from Invitrogen Life Technologies) containing supernatant from the GM-CSF-secreting X63 hybridoma cell line (a gift from Dr. F. Ronchese, Malaghan Institute of Medical Research, Wellington University School of Medicine, Wellington, New Zealand) to give 25 ng/ml GM-CSF. BM cells were cultured in 6-well tissue culture plates (Corning) for 10 days in a humidified incubator at 37°C with 5% v/v CO2. To generate mDC, LPS (from Escherichia coli 026:B6; Sigma-Aldrich) was added to give a final concentration of 1 µg/ml LPS for the final 18 h of culture. On day 10, nonadherent cells were harvested and DC were examined using flow cytometry for purity and expression of DC surface markers.

Enrichment of CL4 CD8+ T cells

Single-cell suspensions were generated from PLNs and spleens from CL4 TCR-transgenic mice. CD8+ CL4 T cells were enriched by positive MACS separation, using anti-CD8 MACS microbeads, together with LS separation columns and Midi-MACS magnets (all obtained from Miltenyi Biotec) according to the manufacturer’s instructions. In some experiments, CL4 CD8+ T cells were labeled with 5 µM CFSE (Molecular Probes) in accordance with previously described protocols (34).

Flow cytometry

Single-cell suspensions were first incubated supernatant from the anti-Fc{gamma}III mAb-secreting 24G2 cell lines followed by incubation with fluorochrome-conjugated mAb against cell surface makers. Anti-CD8, -CD11c, -CD25, -CD40, -CD62L, -CD69, -CD80, -CD86, -CD107a, and -Ly6C mAbs were all purchased from BD Biosciences. Intracellular IFN-{gamma} was detected using a BD Perm/fix kit with GolgiStop (BD Biosciences) according to manufacturer’s instructions and staining with allophycocyanin-conjugated anti-IFN-{gamma} mAb (BD Biosciences). Cells were washed twice in staining buffer and acquired on a FACSCalibur flow cytometer with CellQuest software (BD Cytometry Systems). All flow cytometric analyses were performed using FlowJo software (Tree Star).

Coculture and T cell proliferation

DC were irradiated with 3000 rad and cocultured with CL4 CD8+ T cells in 96-well plates at a ratio of 1:1 in complete RPMI. KdHA peptide, IYSTVASSL (23), was added directly to wells as indicated. In some assays, DC were pretreated with blocking mAb against CD40, CD80, or CD86 (all from BD Biosciences) at a concentration of 25 µg/ml for 1 h before coculture. Cultures were maintained in a humidified incubator at 37°C, 5% v/v CO2 for 72 h, for the final 8 h of culture, 1 µCi/well [3H]thymidine (Amersham Life Sciences). Cells were harvested onto glass fiber filter mats (Cox Scientific) and sealed into sample bags (Wallac-Oy) containing Betaplate Scint (Wallac-Oy). [3H]Thymidine incorporation was determined using a 1450 Microbeta liquid scintillation counter with Microbeta for Windows 2.7 (Wallac-Oy).

Measurement of cytokines

IFN-{gamma} and IL-2 in cell culture supernatants were measured by ELISA according to standard protocols. Briefly, ImmunoSorb plates (Nunc) were coated by overnight incubation with anti-IL-2 or anti-IFN-{gamma} purified capture mAb (BD Biosciences) in carbonate buffer (15 mM Na2CO3, (BDH) 35 mM NaHCO3 (BDH) in double-distilled (dd) H2O). Plates were then washed and blocked with ELISA blocking buffer (PBS supplemented with 1% w/v BSA; Sigma-Aldrich) for 1 h at 37°C. Plates were washed before the addition of cytokine standards (Preprotec) or samples. Plates were incubated in a humidified incubator for 4 h at 37°C before washing. Secondary biotin-conjugated anti-IL-2 or IFN-{gamma} mAb (BD Biosciences) diluted in ELISA blocking buffer was added and plates were incubated for 1 h at room temperature. After further washes, ELISA blocking buffer containing 0.5% v/v ExtrAvidin peroxidase (Sigma-Aldrich) was added to each well and plates were incubated for 30 min at room temperature. Plates were then washed and substrate solution (100 ng/ml tetramethylbenzidine, 0.03% v/v H2O2, 0.05 M Na2HPO4, 0.025 M citric acid (all obtained from Sigma-Aldrich) in ddH2O) was added. Reactions were stopped by adding 100 µl/well 2 M H2SO4 (BDH). Plates were analyzed using a model 3550 microplate reader (Bio-Rad) with Microplate Manager software (Bio-Rad) at a wavelength of 450 nm, with reference to 590 nm.

mRNA for cytokines was measured by the following method: cells were harvested from cocultures and CD8+ T cells were isolated by MACS enrichment as described above. Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) following manufacturer’s instructions. RNA concentration and purity was then determined by spectrophotometry using an Ultraspec 3000 Pro (Pharmacia Biotech). Samples were then standardized by dilution with ddH2O. cDNA was generated from mRNA extracted as above using a SuperScript III First-Strand Synthesis kit (Invitrogen Life Technologies), according to the manufacturer’s instructions.

The following oligonucleotides were purchased from Proligo: IL-4 sense: 5'-ACGGCACAGAGCTATTGATG-3', IL-4 antisense: 5'-ATGGTGGCTCAGTACTACGA-3'; IL-10 sense: 5'-TGCCTTCAGTGAAGAC-3', IL-10 antisense: 5'- AAACTCATTCATGGCC-3'; IFN-{gamma} sense: 5'-AACGCTACACACTGCATCT-3', IFN-{gamma} antisense: 5'-TGCTCATTGTAATGCTTGG-3', hypoxanthine phosphoribosyltransferase (HPRT) sense: 5'-GTTGGATACAGGCCAGACTTTGTTG-3' and HPRT antisense: 5'-GAAGGGTAGGCTGGCCTATAGGCT-3'. PCR amplification consisted of 30 cycles of denaturation at 94°C for 30 s, annealing for 50 s, and extension at 72°C for 45 s. Annealing temperatures used were 53°C for IL-10 and IFN-{gamma}, 60°C for HPRT, and 66°C for IL-4. PCR products were visualized by loading into a 1% w/v agarose (Sigma-Aldrich) gel in Tris-borate-EDTA buffer containing 0.5 µg/ml ethidium bromide (Sigma-Aldrich) which was subjected to electrophoresis before viewing on a UV transilluminator (Uvitec) and photographed.

Cytotoxicity assays

Cytotoxicity was measured by standard chromium release assay. Target P815 cells were labeled by incubation in complete RPMI containing 100 µCi 51Cr/ml, in the form of sodium chromate (Amersham Life Sciences) at 37°C for 2 h together with or without with KdHA peptide. Effector cells were purified from cocultures of CL4 CD8+ T cells and DC using anti-CD8-postive MACS enrichment. Target cells were cultured in 96-well plates with effector cells at an E:T ratio of 1:1 in a humidified incubator at 37°C, 5% v/v CO2 for 16 h. Maximum lysis was determined from mixing labeled target cells with 25% HCL (BDH). Supernatants were mixed with Ultima Gold LLT scintillant fluid (PerkinElmer) at a ratio of 1:3 and scintillation recorded using a 1450 Microbeta liquid scintillation counter with Microbeta for Windows 2.7 (Wallac-Oy). Specific lysis was determined from the following equation: ((experimental counts – spontaneous counts)/(maximum counts – spontaneous counts)) x 100%. CD8+ T cell cytotoxicity was also determined using a flow cytometric analysis of degranulation (63). Briefly, cells were harvested from cocultures or from lymph nodes of recipient mice and incubated with KdHA-pulsed P815 target cells in complete medium containing allophycocyanin-conjugated anti-CD107a mAb (BD Biosciences) at 37°C. After 30 min, monensin-containing GolgiPlug (BD Biosciences) was added according to manufacturer’s instructions. Cells were harvested after a total incubation time of 4 h, incubated with PE-conjugated anti-Thy1.1 mAb (BD Biosciences), and analyzed by flow cytometry.

Assessment of diabetes

Mice in observational groups were tested for the onset of diabetes using Bayer Diastix. Mice diagnosed with glycosuria were retested 2 days later by analysis of venous blood using a Glucotrend 2 blood glucose meter with Accu-Chek glucose strips (Roche Molecular Biochemicals). Diabetes was defined when blood glucose levels were elevated >14 mM/L on two consecutive occasions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Previous studies have demonstrated that naive CD8+ T cells, with specificity for tissue-specific self-Ags, are able to undergo either abortive or productive activation in the tissue draining lymph nodes resulting in either tolerance or autoimmunity respectively (35). It is generally accepted that both responses require BM-derived Ag-presenting DC (11). In this study, we have examined the properties exhibited by DC that are required to evoke these responses.

DC were generated from BM progenitor cells cultured in the presence of GM-CSF. After 10 days, this protocol yielded iDC. DC were matured (mDC) by the addition of bacterial LPS for the final 24 h. Flow cytometric analyses of both iDC and mDC cultures revealed that they were typically composed of >90% CD11c, CD11b double-positive cells. Staining for Gr-1 revealed that there was <5% granulocytes and lack of expression of B220 (CD45R) and CD19, as well as NK1.1, and DX5 indicated that these cultures did not contain any B cells or NK cells (data not shown). Further analyses revealed that both iDC and mDC express equally high levels of MHC class I molecules, whereas expression of the costimulatory molecules: CD80, CD86, and CD40 was greater among mDC than iDC (Fig. 1A). In addition, supernatants from mDC contained high levels of IL-12 whereas supernatants from iDC did not (Fig. 1B).


Figure 1
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  		FIGURE 1. Characterization of iDC vs mDC. iDC were prepared by in vitro culture of BM cells as described. To some cultures, 1 µg/ml LPS was added for the final 24 h to generate mDC. Cells were stained with fluorochrome-conjugated mAbs against CD11c, CD40, CD80, CD86, or MHC class I (H-2Kd). A, Histograms show expression of cell surface markers among CD11c+ cells as follows: iDC (gray lines), mDC (black lines), and control mDC stained with relevant isotype controls (light gray filled). B, Supernatants from DC generation cultures (0 h) and supernatants from DC that had been washed and cultured for 72 h in complete medium (72 h) were assessed for IL-12 production by ELISA. These results are representative of more than three independent experiments.

 
Whether naive CL4 CD8+ T cell interaction with KdHA peptide in vivo results in productive or abortive activation may be determined by examining the profile of expression of cell surface proteins that are associated with T cell activation and differentiation (36). Productive activation of naive CL4 CD8+ T cells in vivo in response to virus infection is characterized by many rounds of division, increased expression of CD69 and CD25, and loss of expression of CD62L (25). Whereas during abortive activation, naive CD8+ T undergoing peripheral tolerance induction divide fewer times and although they express CD69, CD25 is not expressed and CD62L expression remains high (25).

To determine the outcome of CL4 CD8+ T cell interaction with either iDC or mDC in the presence of KdHA peptide in vitro, we first examined the expression of CD69, CD25, and CD62L cell surface proteins. We also examined the level of expression of Ly6C, as there is some evidence that expression of Ly6C may be associated with T cell anergy. Furthermore, a deficiency in Ly6C expression has been linked to autoimmunity (28, 29). MACS-purified naive Thy1.1+ CL4 CD8+ T cells were labeled with CFSE and cocultured in the presence of iDC or mDC pulsed with KdHA peptide. After 72 h, cells were stained with fluorochrome-conjugated Abs and analyzed by flow cytometry. Coculture of CL4 CD8+ T cells with mDC in the presence of 1 µg/ml KdHA resulted in many rounds of cell division as evidenced by the loss of CFSE fluorescence (Fig. 2). Both CD69 and CD25 expression was high among most of the divided cells, and most of the T cells had low expression of both CD62L and Ly6C. Taken together, the data suggest that under these conditions CL4 CD8+ T cells undergo productive activation. When the level of KdHA peptide used to pulse the mDC was reduced by 1000-fold, CL4 CD8+ T cells also underwent the same amount of cell division, as evidenced by loss of CFSE fluorescence, and the profile of cell surface marker expression was the same. However, the level of CD25 expression was not as high as with mDC pulsed with high levels of KdHA peptide, and more of the CL4 CD8+ T cells expressed Ly6C. Similarly, when naive CL4 CD8+ T cells were cocultured with iDC in the presence of 1 µg/ml KdHA peptide, most of the CL4 CD8+ T cells had undergone the same number of cell divisions as with mDC. However, the level of both CD25 and CD69 expression was less among iDC-stimulated cells, with many of the cells not expressing CD69. Also, more CL4 CD8+ T cells expressed higher levels of CD62L and Ly6C, suggesting that fewer cells may be undergoing productive activation. In contrast, when iDC were pulsed with a 1000-fold lower amount of KdHA, the majority of CL4 CD8+ T cells analyzed appeared to be undivided. Importantly, of those cells that had divided, very few cells expressed CD25, and CD62L expression remained high. As for the levels of Ly6C expression, the more the cells divided, the more Ly6C they expressed. Taken together, these results suggest that, when pulsed with low levels of KdHA peptide in vitro, iDC may induce a profile of cell surface expression that is characteristic of abortively activated CL4 CD8+ T cells.


Figure 2
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  		FIGURE 2. CL4 CD8+ T cells activated by iDC in vitro express a partially activated cell surface phenotype. CFSE-labeled Thy1.1+, CL4 CD8+ T cells were cocultured with iDC or mDC in the presence either 1 µg/ml or 1 x 10–3 µg/ml KdHA peptide for 72 h. After harvesting, cells were stained with PE-conjugated anti-Thy1.1 Abs plus fluorochrome-conjugated anti-CD25, -CD69, -CD62L, or -Ly6C Abs. A total of 10,000 Thy1.1+ lymphocytes were collected for each sample. Histograms show expression of activation markers among Thy1.1+ cells vs CFSE fluorescence. These results are representative of more than three independent experiments.

 
The fact that MHC class I expression by both iDC and mDC is approximately equal suggests that the phenotypic differences observed among CL4 CD8+ T cells in response to high and low levels of KdHA peptide is not simply due to a lack of signaling through the TCR (signal 1), induced by iDC when pulsed with low levels of KdHA. Such differences may result from a lack of signaling through costimulatory molecules (signal 2), or cytokines (signal 3). To asses the effect of varying the level of signal 1, T cell proliferation by CL4 CD8+ T cells in response to either iDC or mDC was measured across a wide range of KdHA peptide concentrations. Interestingly, at the maximum concentration of KdHA peptide (1 µg/ml), equally high levels of proliferation were observed among CL4 CD8+ T cells cocultured with either iDC or mDC (Fig. 3A). However, when the concentration of KdHA peptide was reduced, whereas proliferation in the presence of mDC was still readily detectable at 10–9 µg/ml, in the presence of iDC, proliferation did not occur below a peptide concentration of 10–3 µg/ml. These findings suggest that increasing the amount of signal 1 can cause maximal T cell division despite inadequate levels of signal 2 and/or 3.


Figure 3
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  		FIGURE 3. Activation of CL4 CD8+ T cells by iDC vs mDC. CL4 CD8+ T cells were cocultured with iDC or mDC for 72 h in the presence of KdHA peptide at a range of concentrations as shown. [3H]Thymidine was added in excess for the final 8 h of culture. After harvesting, cells were assayed for incorporation of [3H]. A, Proliferation as [3H] incorporation expressed as cpm. Supernatants were harvested from coculture after 72 h and cytokine concentration was measured by standard mAb-capture ELISA. B, IFN-{gamma} concentration; C, IL-2 concentration. Cells from these cocultures were also harvested at 8 and 24 h; CL4 CD8+ T cells were enriched by CD8+ MACS selection, and the presence of cytokine-specific mRNA was determined by RT-PCR. D, The presence of RT-PCR cDNA product for IL-10, IL-4, IFN-{gamma}, and control HPRT amplified from CL4 CD8+ T cells cocultured with either iDC or mDC in the presence of 1 µg/ml, 1 x 10–3 µg/ml, or 1 x 10–5 µg/ml KdHA. The RT-PCR products from Th2-polarized 2G8 cells are shown as a control for IL-10 and IL-4 production. Results are representative of three independent experiments.

 
An important feature of productively activated CL4 CD8+ T cells is that they produce high levels of IFN-{gamma}, whereas CL4 CD8+ T cells undergoing abortive activation do not (25, 37). In the presence of 1 µg/ml KdHA, high levels of both IFN-{gamma} and IL-2 were detected in the supernatant of cocultures of CL4 CD8+ T cells with mDC and the level of both cytokines decreased at lower concentrations of KdHA peptide. However, although there was an equivalent amount of proliferation among CL4 CD8+ T cells at 1 µg/ml KdHA with mDC or iDC, both IFN-{gamma} and IL-2 were barely detectable in supernatants from cocultures containing iDC. When the concentration of KdHA peptide was reduced, despite the fact that T cell proliferation by mDC remained high, no IFN-{gamma} was detected in the supernatants. This indicates that IFN-{gamma} production is not directly coupled to proliferation, suggesting that activation by mDC does not always lead to productive activation.

Activated CD8+ T cells which exhibit a Th1-like cytokine secretion profile have been termed Tc1 cells (38). In addition, it has been shown that activated CD8+ T cells can also differentiate to Tc2 cells, which exhibit a Th2-like cytokine secretion profile (39), secreting IL-4 and secreting a reduced amount of IFN-{gamma} (40). Tc2 cells also have impaired cytotoxic function (41, 42) and may produce IL-10 (36). Analyses of cell lysates from the CL4 CD8+ T cell/DC cocultures revealed presence of mRNA for cytokines associated with Tc1/Tc2 differentiation (Fig. 3D). After 8 h of coculture with mDC pulsed with 1 µg/ml KdHA, lysates of CL4 CD8+ T cells contained IFN-{gamma} mRNA. After 24 h, IFN-{gamma} mRNA could also be detected in lysates from CL4 CD8+ T cells cocultured with mDC pulsed with 10–3 µg/ml KdHA, whereas IFN-{gamma} mRNA was not detected in any of the lysates from the iDC cocultures. Conversely, IL-4 mRNA was detected in lysates of CL4 CD8+ T cells cocultured with iDC and 1 µg/ml KdHA after 8 h of coculture and not in similar mDCs cocultures. Additionally, IL-4 mRNA was detected in lysates from CL4 CD8+ T cell cocultured with iDC pulsed with 10–3 µg/ml KdHA after 24 h. Interestingly, IL-10 was not detected in cell lysates from any coculture tested, suggesting that neither population of DC exhibit immunoregulatory properties. The cytokine data suggest that in the presence KdHA peptide-pulsed mDC, naive CL4 CD8+ T cells exhibit a Tc1-like profile of cytokine production. However, in the presence of peptide-pulsed iDC, CL4 CD8+ T cells exhibit a Tc2-like cytokine profile.

Recent studies have shown that abortive activation which precedes tolerance induction among CL4 CD8+ T cells in vivo is not associated with any CTL effector cell function (43). Therefore, we tested whether the lack of IFN-{gamma} production by CL4 CD8+ T cells cocultured with iDC correlates with a lack of CTL effector function associated with abortive activation. CTL activity among CL4 CD8+ T cells cocultured with KdHA peptide-pulsed iDC or mDC was tested using a standard chromium release assay (Fig. 4A). Coculturing naive CL4 CD8+ T cells with both populations of KdHA peptide-pulsed DC resulted in the generation of CTL. However, the efficiency of target cell lysis was less by CTL generated from the iDC cocultures than by CTL generated from the mDC cocultures, requiring consistently 10-fold more peptide to achieve the equivalent level of target cell lysis. Such reduced killing is consistent with the previous finding that peripheral tolerance induction in InsHA mice is also associated with a reduction in the overall avidity of KdHA-specific CTL (33). Importantly, at the highest level of KdHA peptide, CL4 CTL induced by iDC mediated the level of target cell lysis in vitro as CTL induced by mDC.


Figure 4
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  		FIGURE 4. iDC-activated CL4 CD8+ T cells have impaired cytotoxicity. A, CL4 CD8+ T cells were cocultured with iDC or mDC in the presence of 1 µg/ml KdHA for 72 h. CL4 CD8+ T cells were enriched from these cultures using CD8+ MACS selection and cell concentrations were adjusted to give similar numbers of activated T cells per sample. CL4 CD8+ T cells were incubated at an E:T of 10:1 with KdHA-pulsed P815 target cells that had been labeled with 51Cr. After 16 h, supernatants from these cocultures were harvested and assayed for the amount of 51Cr released using a β scintillation counter. Results are representative of more than three independent experiments. CFSE-labeled CL4 CD8+ T cells were cocultured with iDC or mDC in the presence of KdHA at a range of concentrations as shown. After 72 h, cocultures were incubated with 1 µg/ml KdHA peptide and allophycocyanin-conjugated anti-CD107a Abs for 4 h. Cells were washed and analyzed using flow cytometry. B, Binding of anti-CD107a mAb among represent, mDC-activated (black lines), and iDC-activated (gray lines) CL4 CD8+ T cells. Binding to naive CL4 CD8+ T cells is also shown (solid light gray). Using FlowJo software, the increase in mean fluorescence intensity (MFI) was calculated for divided CFSE-positive CL4 CD8+ T cells relative to control naive CL4 CD8+ T cells. C, Percentage MFI increase by CL4 CD8+ T cells activated by iDC or mDC in the presence of a range of KdHA concentrations. These results are representative of three independent experiments.

 
In Fig. 4A, CTL were generated by coculturing with CL4 CD8+ T cells with DC and 1 µg/ml KdHA peptide. As this level of KdHA induces similar CL4 CD8+ T cell proliferation in the presence of either iDC or mDC (Fig. 3A), it was important to examine the efficiency of the CTL generated by both iDC and mDC with lower levels of KdHA peptide. CTL effector function is associated with the release of lytic granules (44, 45). During this process the lysosome-associated membrane protein-1 (CD107a) become transiently incorporated into the cell membrane (46). Fluorochrome-labeled mAbs against CD107a were used for visualization of CTL functional by flow cytometry (47, 48). When CL4 CD8+ T cells were cocultured with DC in the presence of 1 µg/ml KdHA peptide all cells had increased cell surface recruitment of CD107a (Fig. 4, B and C). However, expression was greater among CL4 CD8+ T cells cocultured with mDC. In the presence of 1 µg/ml KdHA, whereas CL4 CD8+ T cell recruitment of CD107a remained the same among the mDC cocultures, there was a decrease in CD107a recruitment among the iDC cocultures. At 1 x 10–3 µg/ml KdHA, CD107a recruitment decreased further among the iDC cocultures, yet remained high among the mDC cocultures. However, at 1 x 10–5 µg/ml KdHA, CD107a recruitment was the same as the control among CL4 CD8+ T cells from the iDC cocultures, whereas high levels of CD107a were still observed among CL4 CD8+ T cells from the mDC cocultures. These data further support the hypothesis that at very high concentrations of KdHA peptide both mDC and iDC can stimulate the formation of CL4 CTL, whereas at lower concentrations, only mDC have the capacity to induce CTL and iDC induce abortive activation which may result in the induction of tolerance.

Differentiation of CD8+ T cells to IFN-{gamma}-secreting CTL has been previously associated with activation in the presence of IL-12 (49, 50), which provides signal 3 (51). Previous studies, along with experiments detailed in Fig. 1, have indicated that only mDC, and not iDC, produce IL-12 (52). Whether the lack of effector CTL activity among CL4 CD8+ T cells cocultured with iDC is due to lack of IL-12 production was tested by coculturing CL4 CD8+ T cells with either iDC or mDC in the presence of KdHA plus exogenous rIL-12 at the concentration produced by mDC (Fig. 1). Addition of exogenous IL-12 did not affect proliferation of CL4 CD8+ T cells cocultured with iDC or mDC (Fig. 5A). Interestingly, IL-12 addition induced IFN-{gamma} production by CL4 CD8+ T cells cocultured with iDC to similar levels to that observed among mDC cocultures (Fig. 5B). However, the addition of exogenous IL-12 did not restore IL-2 production by iDC cocultures to those levels detected naturally in mDC cultures (Fig. 5C). These results suggest that while IL-12 may be crucial for IFN-{gamma} production, this is not the sole factor that determines abortive vs productive activation of naive CL4 CD8+ T cells.


Figure 5
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  		FIGURE 5. Addition of exogenous IL-12 restores IFN-{gamma} production by iDC-activated CL4 CD8+ T cells, but does not alter proliferation or IL-2 production. CL4 CD8+ T cells were cocultured with iDC or mDC in the presence of KdHA at a range of concentrations as shown. To some cocultures 30 ng/ml IL-12 was also added. [3H]Thymidine was added in excess for the final 8 h of culture. After 72 h, supernatants were removed and cells were harvested and assayed for proliferation by incorporation of [3H]. Cytokine concentrations were measured by standard capture ELISA. A, CL4 CD8+ T cell proliferation expressed as cpm. B and C, Concentrations of IFN-{gamma} and IL-2, respectively. Squares represent cultures containing mDC; circles represent cultures containing iDC. Closed symbols are cultures without the addition of exogenous rIL-12; open symbols are cultures with exogenous IL-12. Results are representative of three independent experiments.

 
We also examined the role of costimulatory molecules expressed by DC (mediators of signal 2), by blocking interactions with CD80, CD86, CD40 either individually or in combination using blocking mAbs in cocultures of CL4 CD8+ T cells with iDC and mDC. Addition of individual anti-CD40, anti-CD80, or anti-CD86 mAbs did not reduce proliferation by CL4 CD8+ T cells cocultured with iDC or mDC in the presence of cognate peptide (Fig. 6A). Proliferation was reduced by a combination of both anti-CD80 and anti-CD86 mAbs, but not by combinations of both anti-CD40 and anti-CD80 or both anti-CD40 and anti-CD86 mAbs. Blocking all three costimulatory molecules together reduced iDC-induced CL4 CD8+ T cell proliferation to background levels, but mDC were still able to induce proliferation (Fig. 6A).


Figure 6
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  		FIGURE 6. Both abortive and productive activation of CL4 CD8+ T cells is dependant on costimulation by CD80 and CD86. iDC or mDC were cocultured for 72 h with CL4 CD8+ T cells in the presence of 1 µg/ml KdHA with or without blocking Abs against one or more of the following costimulatory molecules: CD80, CD86, or CD40. [3H]Thymidine was added in excess for the final 8 h of culture and then cells were assayed for incorporation of [3H]thymidine (A). Results are representative of three independent experiments. iDC or mDC were also cocultured with CL4 CD8+ T cells in the presence of 1 µg/ml KdHA with or without Abs against one or more of the following costimulatory molecules: CD80, CD86, or CD40. After 72 h of coculture, CL4 CD8+ T cells were restimulated with 1 µg/ml KdHA peptide in the presence of allophycocyanin-conjugated anti-CD107a Abs for 4 h. Cells were washed and analyzed using flow cytometry. B, Percentage allophycocyanin-MFI of activated CL4 CD8+ T cells relative to naive CL4 CD8+ T cells. These results are representative of two independent experiments.

 
In addition, we wished to determine whether blocking those signals mediated through the costimulatory molecules CD80 and CD86 (i.e., signal 2) also altered effector function. To this end, we measured CD107a recruitment following reactivation. This allowed us to determine the level of cytolytic function among those CL4 CD8+ T cells that had previously been activated by iDC or mDC in the presence of blocking mAbs against these costimulatory molecules (Fig. 6B). In comparison with controls, there was a reduction in CD107a recruitment among CL4 CD8+ T cells that were initially activated in the presence of anti-CD80 blocking mAb. A greater decrease was also observed with anti-CD86 mAb, and the greatest reduction occurred in the presence of both anti-CD80 and anti-CD86 blocking mAbs (Fig. 6B). Importantly, CD80/CD86 blockade resulted in a reduction in the cytolytic function of iDC-activated CL4 CD8+ T cells to below that of naive CL4 CD8+ T cells. Interestingly, addition of anti-CD40 mAbs increased the level of cytolytic function mediated by iDC-activated CL4 CD8+ T cells, both in the presence or absence of CD80/CD86 blockade. Anti-CD40 mAbs did not appear to alter the cytolytic function of mDC-activated CL4 CD8+ T cells.

The fact that iDC induce abortive activation, while mDCs induce productive activation of CL4 CD8+ T cells is suggested by in vitro phenotypic and functional readouts, which appear to match those of CL4 CD8+ T cells undergoing either tolerance induction or generating autoimmunity in InsHA mice (25). To test directly whether iDC or mDC induce abortive vs productive activation, the ability of peptide-pulsed DCs to activate CL4 CD8+ T cells was examined in vivo. CFSE-labeled CL4 CD8+ T cells were transferred into BALB/c mice. The following day, mice received either iDC or mDC that had been pulsed with either 1 µg/ml or 1 x 10–3 µg/ml KdHA. Seventy-two hours later, CL4 CD8+ T cells isolated from lymph nodes of recipient mice were examined for expression of activation markers and proliferation. As shown in Fig. 7, transfer of KdHA-pulsed iDC did not stimulate up-regulation of CD25, whereas transfer of KdHA-pulsed mDC resulted in the up-regulation of CD25. Additionally, CL4 CD8+ T cells isolated from mice which had received peptide-pulsed iDC had lower levels of CD107a surface recruitment, and fewer donor cells produced IFN-{gamma} upon restimulation with KdHA than for CL4 CD8+ T cells from mice which had received peptide-pulsed mDC. Together, these data suggest that activation of CL4 CD8+ T cells by iDC in vivo is abortive.


Figure 7
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  		FIGURE 7. KdHA peptide-pulsed iDC induce abortive activation of CL4 CD8+ T cells in vivo. A total of 3 x 106 CFSE-labeled, Thy1.1, CL4 CD8+ T cells were injected i.v. into BALB/c mice. The following day, mice were injected i.v. with either mDC or iDC which had been pulsed with either 1 µg/ml or 1 x 10–3 µg/ml KdHA peptide. Seventy-two hours later, lymph node cells were harvested and stained with PE-conjugated anti-Thy1.1 mAbs and anti-CD25 mAbs (A) and then restimulated for 4 h in the presence of monensin before intracellular staining with anti-IFN-{gamma} mAb (B) or were restimulated for 4 h in the presence of anti-CD107a mAb (C). Results are representative of three independent experiments.

 
The fact that iDC were able to induce CL4 CD8+ T cell division in vivo prompted us to test whether such activation was associated with the formation of effector CTL or with the induction of peripheral tolerance. To this end, CL4 CD8+ T cells were activated in vivo by adoptive transfer of KdHA-pulsed iDC or mDC into InsHA mice. Were CL4 CD8+ T cells to undergo productive activation to form anti-HA CTL, then one would anticipate the destruction of HA-expressing pancreatic islet β cells, leading to autoimmune diabetes. Thus, recipient mice were monitored for diabetes for up to 6 wk after transfer. As shown in Table I, transfer of mDC-pulsed with 1 µg/ml KdHA peptide into InsHA mice that were given naive CL4 CD8+ T cells resulted in diabetes within 3 wk, whereas transfer of mDC pulsed with 1 x 10–3 µg/ml KdHA peptide failed to induce diabetes (Table I), despite the fact these mDC were able to induce CL4 CD8+ T cell proliferation and IFN-{gamma} production in vivo (Fig. 7). Critically, transfer of iDC-pulsed with either 1 or 1 x 10–3 µg/ml KdHA peptide did not generate autoimmune diabetes. However, when iDC were pulsed with excess KdHA peptide at 1 mg/ml then all mice developed diabetes, clearly demonstrating that iDC are able to prime CTL capable of mediating autoimmune diabetes. In addition, control mice which had not received CL4 CD8+ T cells also did not develop autoimmune diabetes (data not shown), indicating that diabetes only occurs as a result of cognate Ag-specific activation of naive CL4 CD8+ T cells by iDC.


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  		Table I. Activation of CL4 CD8+ T cells in vivoa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies have demonstrated that, as a result of encountering KdHA epitopes expressed in the PLN of InsHA mice, CL4 CD8+ T cells undergo abortive activation which results in the induction of self-tolerance (33); whereas CL4 CD8+ T cells activated by KdHA epitopes during PR8 infection of InsHA recipients mount productive responses that result in autoimmune diabetes (25). Therefore, although all CL4 CD8+ T cells express the same TCR at the same level with the same high avidity for KdHA peptide, the context in which they encounter KdHA Ag determines their functional response. Several studies have shown that encounter with self-Ags presented by iDC results in peripheral tolerance induction among T cells (53), whereas Ag presentation by mDC induces T cell priming (15). In this study, we have examined the consequences of naive CD8+ T cell activation by the same self-epitopes presented either by iDC or mDC in vitro and in vivo.

T cell activation by APC is driven by a combination of at least three signals: signal 1 is mediated by TCR interactions with cognate peptide/MHC; signal 2 is mediated by interactions between cell surface costimulatory molecules expressed by APCs with ligands on T cells; and signal 3 is mediated by secretory factors including cytokines. iDC and mDC differ in their ability to provide mediators for both signal 2 and 3. Our results show that there is a reduction in proliferation of naive CL4 CD8+ T cells, both in total numbers of responding cells, and in the number of rounds of cell division when activated by iDC compared with activation by mDC. Moreover, iDC needed to be pulsed with at least 10,000-fold more KdHA peptide than mDC, to generate similar levels of CL4 T cell proliferation. Although it has been suggested that CD28 costimulation through CD80/CD86 molecules is required to generate effector CTL responses (54, 55), our data indicate that iDC, which express lower levels of cell surface CD80/CD86 costimulatory molecules than mDC, are able to drive CL4 CD8+ T cell proliferation to the same extent as mDC, providing that sufficient levels of signal 1 are supplied. Although previous studies have shown that high doses of cognate Ag may be able to rescue CD8+ T cells activated in the absence of costimulation (56, 57, 58, 59), under these conditions, both CD80/CD86 costimulation was required to induce full CTL effector function.

When naive CL4 CD8+ T cells were activated by mDC in vitro and in vivo they expressed a cell surface phenotype typical of the productively activated effector CTL which are generated as a result of influenza virus infection of recipient host mice (25), whereas when CL4 CD8+ T cells were activated in the presence of iDC, they expressed only a partially activated cell surface phenotype that was similar to CL4 CD8+ T cells undergoing peripheral tolerance induction by abortive activation in vivo (25). Interestingly, CL4 CD8+ T cells activated by iDC differed from those activated by mDC in their patterns of CD25 and Ly6C cell surface expression in vitro and in vivo. Ly6C expression has been previously associated with productively activated T cells, memory T cells, and effector CTL (27). However, our studies reveal that Ly6C expression is associated with impaired proliferation and lack of differentiation into effector CTL following encounter with peptide-pulsed iDC. Other studies have demonstrated that Ly6C expression may also be associated with reduced IL-2 production among CD4+ T cells (28), which as a result, may affect the expression of CD25. Further studies have revealed that there is a deficiency in Ly6C expression among autoimmune susceptible mouse strains such as NOD and NZB (29), suggesting that low levels of Ly6C-expressing CD8+ T cells may also be linked to failure of self-tolerance induction. However, the precise role of Ly6C still remains to be determined.

Encounter with KdHA-pulsed iDC did not result in IFN-{gamma} production by CL4 CD8+ T cells in vitro and in vivo, indicating that they fail to differentiate into effector CTL. Additionally, IFN-{gamma} and IL-2 mRNA production was not observed among these CL4 T cells in vitro, whereas IL-4 mRNA was detected. IL-4 production by CD8+ T cells has previously been associated with a low efficiency in cytotoxicity (41).

We demonstrated that CTL could be generated when CL4 CD8+ T cells were stimulated by either iDC or mDC pulsed with high levels of KdHA peptide in vitro. However, the CTL generated by iDC had a reduced capacity to kill target cells compared with mDC-stimulated CTL. Furthermore, at low levels of cognate peptide, only mDC were able to generate CTL in vitro. Critically, when pulsed with high levels of KdHA peptide, only mDC were able to induce CTL capable of inducing autoimmune diabetes, and only when pulsed with an excess amount of cognate peptide (1 mg/ml) were iDC able to induce autoreactive CTL in vivo. We suggest that when only low levels of signals 2 and 3 are provided, which is the case with iDC, then the level of self-peptides presented is key in determining whether naive CD8+ T cell responses result in self-tolerance or autoimmunity. This is particularly important during situations whereby increased levels of self-Ags may be made available during persistent virus infections.

To understand the mechanisms by which iDC may induce self-tolerance among CD8+ T cells, we examined the importance of signals 2 and 3 in "abortive" vs "productive" activation. IL-12 is a mediator of signal 3 and its production by mDC is likely to control IFN-{gamma} production by CL4 CD8+ T cells. Indeed, addition of exogenous IL-12 to CL4 CD8+ T cells cocultured with iDC resulted in IFN-{gamma} production to similar levels seen among coculture with mDC. However, addition of exogenous IL-12 to iDC cocultured did not restore proliferative responses of CL4 T cells to a level generated by mDC. Rather, it appeared that proliferation by CL4 T cells, abortively activated by iDC, relied upon signal 2 which was provided by coexpression of both CD80 and CD86. It is not surprising that addition of exogenous IL-12 initiated IFN-{gamma} production by CL4 CD8+ T cells activated by iDC. It was previously shown that IL-12 polarizes T cell subsets toward a Tc1/Th1 phenotype (49, 50). However, on its own, IL-12 does not appear to restore productive activation to CL4 CD8+ T cells stimulated by iDC. These data show that, even when the levels of signal 1 and 3 provided by either iDC or mDC are the same, differences in activation of CL4 CD8+ T cells by iDC vs mDC are still observed.

Our studies using blocking mAbs indicated an essential role for the costimulatory molecules CD80 and CD86 in generating effector CTL function. The fact that proliferation and cytolytic function, mediated by mDC, is not completely blocked by addition of mAbs against CD80 and CD86 indicates that costimulation may be provided by alternative ligands expressed by mDC. It is likely that CD40 engagement licenses DC maturation and thus increases CD80/CD86 expression resulting in an increase in signal 2. Therefore, CD80 and CD86 may be crucial for the activation of CL4 CD8+ T cells by iDC. In contrast, mDC activation of CL4 CD8+ T cells may also rely on other costimulatory molecules not tested in this study; as in our study, CL4 proliferation and the generation of CTL still occurred despite the presence of blocking mAbs against CD40, CD80, and CD86.

Presentation of KdHA epitopes by mDC in InsHA mice results in the productive activation of naive CL4 CD8+ T cells, generating CTL effector function and autoimmunity. Although presentation of KdHA by iDC also results in CL4 CD8+ T cell activation and proliferation, such activation does not generate CTL effector function or autoimmunity unless they are saturated with an excess level of cognate peptide. Thus under standard conditions, activation by iDC in vivo is generally abortive; therefore suggesting that iDC induce and maintain the steady state of peripheral tolerance. However, we have clearly demonstrated that under certain conditions, iDC can also initiate productive T cells responses both in vitro and in vivo. Therefore, we further suggest that abortive activation is not be the only possible outcome of activation of CD8+ T cells by iDC in vivo.

To explain our data, we propose a paradigm whereby naive CD8+ T cell activation results from a combination of signals 1, 2, and 3. Alterations in the levels of these signals produce responses ranging from no activation through abortive activation (including peripheral deletion, anergy induction, and acquisition of regulatory properties), to productive activation and the formation of CTL. This paradigm is in contrast to an established model where a naive CD8+ T cell differentiation into CTL occurs once a threshold level of signaling is reached. According to this established model, the induction of anergy and the subsequent failure of CD8+ T cells to respond is a passive process that occurs when this threshold is not reached.

Our alternative model suggests that DC maintain the steady state of self-tolerance in an active and dynamic manner which allows the initiation of productive immune responses against tissues when necessary. For example, when a mutated cell overexpresses a self-Ag during tumor development, a productive immune response to the self-Ag is highly desirable (33, 60). Another situation when CTL responses against host cells are desirable occurs during virus infection. To prime effective antiviral CTL, DC must cross-present virus peptides that are derived from infected host cells. However, as these DC which also cross-present self-Ags derived from infected host cells are mature (4, 5), they often generate concomitant autoimmunity (61). Nevertheless, when autoimmunity is observed, it is generally transient and localized, and the steady state of self-tolerance is restored following removal of the virus. Such transient autoimmunity is therefore likely to develop as a consequence of increasing the levels of signals 1, 2, or 3 supplied by the DC under inflammatory conditions, thus diverting abortive activation to productive activation. However, when inflammation subsides following virus clearance, the levels of signals 1, 2, and 3 are again altered and self-tolerance is restored.

However, some individuals do develop sustained autoimmunity which may arise following increases in signals mediated by mDC under inflammatory conditions, or when the amount of self-Ag that is cross-presented provides maximal levels of signal 1 by iDC. For example, the onset of type 1 autoimmune diabetes has been associated with a preceding viral infection and/or physical trauma to the pancreas (62). It is possible that, during these types of situations, autoimmunity may arise because of the fact that increases in DC-mediated signaling are either too great or are too prolonged. Therefore, the productive activation of self-reactive T cells cannot be diverted to abortive activation before excessive tissue destruction occurs which would release large amounts of self-Ags and provide maximal levels of signal 1.


   Acknowledgments
 
We thank Dr. Lindsay Nicholson for critical advice in preparing this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Juvenile Diabetes Research Foundation Grant 1-2000-99 (to D.J.M.). B.J.E.R. was in receipt of Medical Research Council U.K. Studentship G78/7503. Back

2 Address correspondence and reprint requests to Dr. David J. Morgan, Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, U.K. Back

3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, mature DC; BM, bone marrow; HA, hemagglutinin; dd, double distilled; HPRT, hypoxanthine phosphoribosyltransferase; PLN, pancreatic lymph node. Back

Received for publication October 11, 2006. Accepted for publication June 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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