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International Immunology Advance Access originally published online on December 16, 2005
International Immunology 2006 18(1):199-209; doi:10.1093/intimm/dxh363
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Presentation of self-antigens on MHC class II molecules during dendritic cell maturation

Heather L. Zwickey1,*, Julia J. Unternaehrer2,* and Ira Mellman2

1 Helfgott Research Institute, National College of Naturopathic Medicine, 049 SW Porter Street, Portland, OR 97201, USA
2 Department of Cell Biology and Section of Immunobiology, Ludwig Institute for Cancer Research, Yale University School of Medicine, 333 Cedar Street, PO Box 208002, New Haven, CT 06520, USA

Correspondence to: I. Mellman; E-mail: ira.mellman{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary material
 References
 
Little is known about how dendritic cells (DCs) maintain a balance between tolerance and immunity for antigens synthesized by DCs themselves. Using transgenic DCs expressing a model self-antigen, in vitro self-peptide–MHC class II complex formation and presentation increased with DC maturation, as for exogenous antigens. In vivo, however, even ‘immature’ DCs isolated from steady-state lymph nodes expressed MHC at mature cell levels, although many were also CD86 low. Adoptive transfer of naive specific T cells into unstimulated transgenic mice resulted in tolerance. If the mice were also injected with anti-CD40 or Listeria monocytogenes, there was robust specific T cell expansion and inflammation. Thus, DC-endogenous antigens may induce tolerance, but only in the absence of potent maturation stimuli.

Keywords: antigen competition, antigen presentation, CD4 T cells, co-stimulation, dendritic cells, HEL, self-antigen, tolerance


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary material
 References
 
The immune system must maintain a balance between responding to foreign antigens and being tolerant to self-antigens. Dendritic cells (DCs) must play a role in maintaining this balance since they exhibit the paradoxical capacity to stimulate naive T cells to respond to foreign antigens and also to induce T cell tolerance to self-antigens (1, 2). During infection, DCs can present an array of microbial antigens, environmental antigens and self-antigens. How DCs selectively activate immune responses without simultaneously inducing autoimmunity remains poorly understood. One possibility is that lymph node (LN) DCs in uninfected animals define immunologic self and effectively tolerize the T cell repertoire. Although appealing, current understanding of DC cell biology would appear to suggest that this mechanism may not be quite so simple.

Based largely on studies performed in vitro, it is clear that DCs regulate their capacity for antigen processing and presentation by a terminal differentiation program termed maturation, an event triggered by a variety of microbial and inflammatory stimuli (3, 4). Maturation results in the proteolysis of internalized antigen and the enhanced formation of MHC class II–peptide complexes that traffic from lysosomal compartments to the cell surface (5–10). Formation of MHC class I–peptide complexes derived from exogenous antigens is also enhanced by maturation (11, 12). Importantly, mature DCs also exhibit dramatic increases in the surface expression of co-stimulatory molecules such as CD86, required for the activation of naive T cells.

In contrast, T cell tolerance is thought to occur when antigens are presented by DCs in the absence of co-stimulation (13–15). Since DCs expressing low levels of co-stimulatory molecules would, on the basis of definitions established in vitro, be considered as immature, there is an apparent contradiction: immature DCs are less efficient at generating peptide–MHC complexes than mature DCs, at least in the case of MHC class II. While MHC class I systems have demonstrated the ability of DCs to break tolerance (16), DCs, due to their phagocytic capabilities, are most adapted to presenting antigens in MHC class II. One possibility is that the inefficient processing and presentation activity of immature DCs is sufficient to ensure peripheral tolerance. Alternatively, current definitions of immature versus mature DCs, defined in vitro, may be insufficient to explain this aspect of DC function. Indeed, recent in vivo evidence has demonstrated that DCs can present exogenously administered antigens in the absence of a maturation stimulus, a condition that induced a tolerogenic response (17–19). For example, when hen egg lysozyme (HEL) was targeted with anti-DEC205 to DCs in mice transgenic for HEL-specific TCRs, T cells exhibited a strong but transient response and then became unresponsive to subsequent administrations of antigen (17, 18, 20). However, when a maturation signal (anti-CD40) was injected along with the antigen, a sustained immunogenic T cell response was observed. Thus, antigen could be presented in vivo by LN DCs at the steady state, with immunological outcome being determined by the presence or absence of a maturation stimulus. The steady-state DCs were immature with respect to their inability to stimulate an immunogenic response, but they were mature in their ability to present antigen, and to express at least moderate levels of CD86 (21).

In an effort to address the apparent discrepancy between the phenotypic and functional features of immature versus mature DCs, we have examined the processing and presentation of a surrogate self-antigen, transgenic HEL, expressed by the DCs themselves. Very recent studies using similar mice demonstrate that tissue DCs from HEL- or ovalbumin peptide-expressing mice are able to process and present endogenous antigen (22, 23). Using functional T cell assays as well as a specific mAb to an HEL–MHC class II peptide complex, we directly compared the ability of the HEL transgenic DCs in vitro and in LN to form peptide–MHC complexes, stimulate T cells and regulate T cell outcome in response to maturation stimuli.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary material
 References
 
Mice
Membrane hen egg lysozyme (mHEL) and soluble hen egg lysozyme (sHEL) mice were derived by breeding C57BL/6-TgN(KLK4mHEL)6Ccg and C57BL/6-TgN(ML5sHEL)5Ccg 4- to 8-week old male mice (24) with C3H/HeJ 4- to 8-week old female mice purchased from the Jackson Laboratory (Bar Harbor, ME, USA), respectively. Non-HEL transgenic control mice were derived from crossing C57BL/6 mice with C3H/HeJ mice. 3A9 TCR transgenic mice (TCR specific for HEL–I-Ak complex) were provided by Michel Nuessenzweig (Rockefeller University). AND mice [TCR specific for MCC/pigeon cytochrome C (PCC)–I-Ek] were provided by Kim Bottomly (Yale University).

Antibodies
Anti-CD4–FITC, CD4–PE, CD11c–cytochrome, CD45Rb–PE, CD86–PE, Vß8–PE, I-Ak–FITC, I-Ek–FITC, anti-CD40 (3/23) and I-Ab–FITC, as well as isotype control IgG2b were purchased from BD PharMingen (San Diego, CA, USA). Secondary antibodies including goat anti-rat 488 were purchased from Molecular Probes (Eugene, OR, USA). Three-color flow cytometry was performed on a FACSCaliburTM flow cytometer and analyzed using FlowjoTM or CELLQuestTM software (Becton Dickinson).

Cell preparation and analysis
DCs were derived from bone marrow as described (25). The progenitors were cultured in supernatant harvested from granulocyte macrophage colony-stimulating factor-transfected J558L cells, in 24-well plates (Costar Corp, Cambridge, MA, USA). Immature DCs were harvested on day 5 of culture. Alternatively, immature DCs were stimulated to mature with LPS (Escherichia coli 0111:B4, 50 ng ml–1; Sigma Chemical Co., St Louis, MO, USA). Mature DCs were harvested 48 h post-stimulation.

CD4 T cells were isolated from mechanically disrupted LNs and spleens of TCR transgenic mice. RBCs were lysed with ammonium chloride. Naive T cells were isolated using negative depletion. CD4 T cells were incubated with anti-CD8 (TIB105), FcR (24G2), pre-B cell marker (TIB164) and I-A (212.A1) for 30 min on ice. Cells were washed, and rat anti-mouse IgG, goat anti-rat IgG, and goat anti-mouse IgM were added to the cells for 45 min on ice. Cell suspensions were then placed on magnet to negatively select for CD4 T cells. Naive CD4 T cells were positively selected by use of anti-L-selectin (MEL-14) provided by Kim Bottomly's laboratory (Yale University).

Antigen presentation assays
Immature or mature DCs were fixed in 1.0% PFA for 10 min at room temperature and then washed extensively. DCs were added to 96-well round-bottom plates at 5 x 104 DCs per well. Primary T cells from TCR transgenic mice were added to DCs in triplicate at ratios of 20:1, 10:1, 5:1 and 1:1. Unless stated, data shown are from DC–T cell ratio of 1:1. Supernatant was collected and frozen after 24 h. IL-2 was analyzed by ELISA.

Antigenic conditions
Artificial co-stimulation.
The 96-well plate was pre-coated with 40 ng ml–1 anti-CD28 (BD PharMingen) at 4°C overnight. The plate was washed with PBS and the antigen presentation assay was completed as described above.

Protein and peptide.
HEL was obtained from Sigma Aldrich St Louis, MO, USA). Protein was dissolved in PBS at a concentration of 20 mg ml–1. LPS was removed with Kuttsuclean (Maruha Corporation, Ibaraki, Japan). Aliquots of HEL peptide were stored at –20°C. Protein was added to DC cultures at graded doses as described. Mouse lysozyme peptide (RGDQSTDYGIFQINSR) was synthesized by Keck Foundation Biotechnology Resource Laboratory (New Haven, CT, USA). Desiccated peptide was dissolved in PBS at a concentration of 50 mg ml–1. Peptide was added to DC culture at graded doses. PCC protein was obtained from Sigma Aldrich. LPS was removed from PCC with Kuttsuclean.

Necrotic DCs.
Mature bone marrow-derived DCs were generated from PCC transgenic mice (26). DCs were harvested on day 7, and frozen at –80°C for 1 h. They were allowed to thaw at room temperature for 15 min, and then frozen again at –80°C for 1 h. After the second thaw, cells were tested for death by propidium iodide staining and trypan blue. Dead cells were added to day 5 DC cultures at graded doses.

Infection.
Escherichia coli DH5{alpha} was grown in LB overnight and the resultant culture was determined to contain ~108 ml–1 bacteria by plating. Listeria monocytogenes was grown in TPB overnight, and the resultant culture was determined to contain ~8 x 107 bacteria per milliliter. Bacteria were washed in PBS three times, and added to DCs at graded doses. DCs were infected in antibiotic-free RPMI containing 5% FCS and 1% mouse serum for 6 h. After 6 h, infected DCs were washed in PBS, re-suspended in RPMI supplemented with penicillin, streptomycin and gentamicin and co-cultured with T cells for antigen presentation assay.

CD11c separation
Axillary, brachial, inguinal, salivary and mesenteric LNs and spleens were harvested on ice. To isolate splenic and LN DCs, spleen or LNs were digested in 400 U ml–1 collagenase (Sigma) or Liberase Blendzyme 2 (Roche) for 20 min on ice. Organs were teased apart with 19-g needles, and pushed through a 70-µm cell strainer. The resultant cell suspension was incubated with anti-FCR for 15 min, and then incubated with CD11c magnetic microbeads (Miltenyi, Auburn, CA, USA). CD11c-positive cells were isolated by magnetic separation as described (Miltenyi protocol). In some experiments, two consecutive separations were performed for each sample to increase purity of CD11c+ or CD11c– cells.

Adoptive transfer and in vivo DC maturation
T cells were harvested from TCR transgenic mice as described above. T cells were labeled with 2 mM CFSE (Sigma Aldrich) for 20 min at 37°C, washed and re-suspended in PBS at 108 ml–1. Recipient mice were anesthetized with methoxyflurane (Medical Developments, Springvale, Australia). Fifty microliters of T cells (5 x 106 per mouse) was injected intravenously (i.v.) (retro-orbital) into 8- to 12-week old gender-matched mice. Spleens and LNs were harvested at various times post-transfer. T cells were stained for anti-CD4–cytochrome, anti-Vb8–PE, and analyzed for CFSE expression by flow cytometry. Alternatively, mice were injected with CFSE-labeled T cells on day 1, and injected intraperitoneally (i.p.) with either L. monocytogenes (103) or anti-CD40 (90 µg; clone 3/23 from BD PharMingen) 24 h later. Mice were sacrificed at 4 days and 8 days post-transfer. T cells were harvested and stained as described above. For in vivo DC maturation studies, mice were injected i.p. with PBS or anti-CD40 (FGK4.5, gift of Bottomly's laboratory). Forty-eight hours later, LNs were harvested and CD11c+ cells isolated as above.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary material
 References
 
Immature DCs can present endogenous HEL
Although it is clear that both in vitro and in vivo, DCs can present self-antigens following the phagocytosis of apoptotic cells (27), very little is known about the processing and presentation of self-antigens expressed by the DCs themselves. To this end, we analyzed DCs isolated from the bone marrow of two different HEL transgenic mouse strains (28). The first expressed a membrane-bound form of HEL (mHEL) expressed under the control of a MHC class I promoter and the second expressed a secreted HEL construct (sHEL) under the control of a metallothionein promoter. Thus, many cell types in these mice, in addition to DCs, would be expected to express HEL as self-protein. These mice were originally created as a model for B cell tolerance (24, 29). Because most of the antibodies and T cell reagents used to detect HEL processing and presentation have been developed for HEL–I-Ak MHC II (30, 31), the H-2b HEL transgenic mice were crossed with the C3H/HeJ (H-2k). The F1 generation was used to assess whether DCs present HEL as self-peptide bound to I-Ak.

We first examined the ability of immature and mature HEL transgenic DCs to generate HEL peptide–MHC II in vitro using T cells specific for the 46-61 HEL peptide–I-Ak complex. As shown in Fig. 1, immature DCs from mHEL and sHEL transgenic mice, or from non-transgenic mice, were unable to stimulate IL-2 release by the T cells (white bars). LPS-matured mHEL and sHEL DCs were, as expected, efficient at T cell stimulation (black bars). To determine if the low level of T cell stimulation by HEL transgenic DCs was due to an absence of peptide–MHC II complexes or the lack of co-stimulatory molecules required for naive T cell activation, we next provided T cells with artificial co-stimulation (anti-CD28) during the antigen presentation assay. Under these conditions, immature mHEL and, to a lesser extent, sHEL DCs were capable of stimulating IL-2 release from naive 3A9 T cells (Fig. 1, gray bars). Anti-CD28 had little effect when added to assays containing non-HEL transgenic DCs. Thus, immature DCs express sufficient HEL peptide–MHC II complex at least when exogenous co-stimulation is supplied.



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  		Fig. 1. Self-MHC–peptide complexes on mature DCs stimulate naive T cells. Immature (day 5) DCs (white bars) were harvested, fixed and cultured with naive T cells from TCR transgenic mice (3A9). Mature DCs (black bars) were generated by adding LPS (50 ng ml–1) to immature DCs on day 5. Mature DCs were harvested on day 7, fixed and cultured with naive T cells from TCR transgenic mice. After 24 h, supernatants from DC–T cell co-cultures were harvested and analyzed for IL-2 production by ELISA. Artificial co-stimulation with anti-CD28 (gray bars) was achieved by culturing fixed immature DCs with T cells in a plate pre-coated with anti-CD28 (40 ng ml–1 for 2 h, washed three times with PBS).

 
Foreign antigen does not compete with self-antigen for presentation
Although presentation of an endogenous self-antigen by immature DCs might contribute to peripheral tolerance, presentation by mature DCs that also express co-stimulatory molecules might have the effect of triggering autoimmune responses. It is often thought that presentation of self by mature DCs is limited by competition for MHC molecules by excess foreign antigen. To test this possibility, HEL transgenic DCs were fed recombinant PCC and LPS. T cells specific for PCC were used to measure PCC peptide–MHC II while T cells specific for HEL were used to measure simultaneous presentation of HEL. As shown in Fig. 2(A), increasing amounts of PCC added to the medium did not efficiently compete the presentation of endogenous mHEL; only at the highest antigen dose was a modest reduction seen. At the same time, PCC peptide was itself efficiently presented. This suggests that foreign antigen does not effectively compete with self-antigen in mature DCs.



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  		Fig. 2. Exogenous antigen does not efficiently compete with self-antigen for MHC molecules. Exogenous antigen in the form of PCC protein (A) or necrotic PCC transgenic DCs expressing PCC on their surface (B), along with LPS, were added to DCs on day 5 of culture. DCs were harvested at day 7 and co-cultured with HEL-specific T cells (left panels) or PCC-specific T cells (right panels). A DC to CD4 T cell ratio of 1:1 is shown. (C) Bacterial infection does not significantly reduce the amount of self-antigen presented by DCs. Immature (day 5) DCs were infected with Escherichia coli DH5{alpha} (left panel) or Listeria monocytogenes (right panel) in antibiotic-free media for 6 h. Following infection, DCs were washed three times in PBS, and co-cultured with T cells in antibiotic containing media. After 24 h, supernatants from DC–T cell co-cultures were harvested and analyzed for IL-2 production by ELISA.

 
To test whether the same phenomenon holds true for other antigens, mHEL or sHEL transgenic DCs were pulsed with necrotic PCC transgenic DCs that express PCC on their surface (26). Not only PCC but also an array of potential antigens from the necrotic cells might compete with HEL for MHC II. As shown in Fig. 2(B), however, increasing doses of necrotic cells did not reduce the expression of HEL or PCC peptide–MHC II.

Perhaps the most physiological exogenous antigen that DCs encounter in vivo is bacteria. Therefore, HEL transgenic DCs were infected with live E. coli or L. monocytogenes to determine whether bacterial antigens compete for MHC II. Six hours after infection, DCs were assayed for presentation to 3A9 T cells. Antigen from E. coli did not effectively compete with HEL in mHEL transgenic DCs; at best, there was a slight decrease in presentation at the highest multiplicities of infection, particularly in the case of the sHEL trangenics (Fig. 2C). Very similar results were obtained for L. monocytogenes.

Although reagents were not available to allow monitoring of the formation of peptide–MHC II complexes from bacterial antigens, it seems likely that (as found for PCC) immunogenic complexes containing bacterial peptides did form. That bacterial antigens did not compete with endogenous self-antigens is likely to reflect the fact that mature DCs express large quantities of MHC II; there may well be more than a sufficient amount to accommodate both endogenous and foreign peptides. Thus, mature DCs can stimulate both autoreactive and foreign T cell responses simultaneously because they present endogenous self-peptides even in the presence of excess foreign antigens.

CD11c+ mHEL transgenic DCs express self-peptide–MHC II in vivo
To determine if HEL transgenic DCs in vivo expressed the HEL peptide–MHC II, DCs were isolated from LNs and spleens using magnetic beads coated with anti-CD11c and analyzed immediately. As shown in Fig. 3(A), CD11c+ LN DCs from mHEL mice stimulated naive 3A9 T cells in vitro in the absence of anti-CD28. Similar results were obtained for splenic DCs (data not shown). Thus, even at steady state, LN DCs expressed the endogenous self-peptide–MHC II complex. By this criterion, these LN DCs would be defined as being functionally mature.



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  		Fig. 3. CD11c+ cells isolated from LNs express more HEL–I-Ak than CD11c– cells. LNs were harvested from HEL transgenic or non-transgenic mice on ice. LN cells were separated by CD11c magnetic column at 4°C. CD11c+ population was further purified by separation on a second CD11c magnetic column. The CD11c+/– cells were then co-cultured with HEL-specific T cells. After 24 h, supernatants from DC–T cell co-cultures were harvested and analyzed for IL-2 production by ELISA.

 
Controlled expansion of HEL-specific T cells transferred into HEL transgenic mice
The presence of functionally mature DCs expressing HEL-peptide–MHC II in vivo found to be stimulatory for T cells in vitro (i.e. functionally mature) was surprising since the presence of such cells in principle would create an opportunity for autoimmunity. Because these mice were transgenic for HEL since birth, however, neonatal or ‘central tolerance’ might have already deleted all such autoreactive T cells. We therefore decided to study how adoptively transferred autoreactive T cells responded to self-antigen presentation in vivo in these mice. HEL-specific 3A9 T cells were pre-labeled with CFSE and i.v. transferred into the mHEL transgenic mice or non-transgenic controls. These recipient mice are not TCR transgenic, thus 3A9 TCR transgenic T cells supplement an otherwise wild-type T cell repertoire. All CD4+ T cells were re-isolated at different time points and analyzed for proliferation by FACS.

HEL TCR transgenic T cells transferred into mHEL transgenic mice had expanded significantly at 2 days post-transfer (Fig. 4A), supporting the observations of Huang et al. (32). Although fewer than 30% of the cells had divided after transfer into non-transgenic animals, 70% of the T cells in mHEL mice had divided and nearly 40% had undergone at least four cell divisions. Two-thirds of the CD4+ T cells recovered from HEL transgenic mice were TCR transgenic, while one-third of those retrieved from non-HEL transgenics carried the 3A9 transgene.



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  		Fig. 4. HEL-specific T cells divide in vivo when transferred into HEL transgenic mice. (A) Naive HEL transgenic T cells were harvested from female 3A9 TCR transgenic mice. T cells were labeled with CFSE. Cells (5 x 106) were injected i.v. into 12-week-old female HEL transgenic or non-transgenic mice. T cells were harvested from spleen and LNs at designated time points. LN cells were pooled from each group and CFSE was analyzed on CD4+ cells (gated) to detect in vivo cell division. (B) HEL-specific T cells are refractory to further stimulation after transfer into mHEL/F1 transgenic mice. Naive HEL transgenic T cells were harvested from female 3A9 transgenic mice. Unlabeled cells (5 x 106) were injected i.v. into 12-week-old female HEL transgenic or non-transgenic mice. T cells were harvested from spleen and LNs at designated time points. T cells were then labeled with CFSE and cultured with non-HEL transgenic DCs fed 1 mg ml–1 HEL. T cells were harvested, gated and analyzed for their ability to divide. (C) DCs derived from mHEL transgenic mice were matured by physical disruption and co-cultured with 3A9 T cells adoptively transferred and re-isolated from mHEL transgenics or from non-transgenics. Supernatants from the T cell–DC co-cultures were collected and analyzed for IL-2 production as described above.

 
We next tested whether T cells that had been adoptively transferred into the HEL transgenic mice were still responsive to HEL in vitro. The T cells recovered from these animals also appeared to have become tolerant to HEL (Fig. 4B). When incubated together with peptide-loaded DCs in vitro, T cells adoptively transferred and re-isolated from mHEL transgenics and then CFSE labeled were largely refractory, with 83% failing to exhibit a single cell division; only 7% had clearly divided. These T cells were also unable to produce IL-2 in response to antigen (Fig. 4C), even though two-thirds of the recovered T cells were TCR transgenic. This was in marked contrast to T cells re-isolated from non-HEL transgenic animals, where at least half were stimulated to proliferate. The HEL-specific response is diluted by the presence of a polyclonal T cell repertoire from the recipient mice. Thus, at the steady state, adoptively transferred antigen-specific T cells appeared to have been tolerized by the HEL transgene.

Consistent with the induction of tolerance, few, if any, of the HEL transgenic mice were found to develop any manifestation of inflammation following adoptive transfer. Only one in eight exhibited skin lesions or chronic (>2 weeks) splenomegaly. The rest of the mice survived without incident.

Thus, the large majority of mice did not develop inflammation despite the fact that autoreactive T cells had been transferred into animals containing LN DCs [and other antigen-presenting cells (APCs)] expressing self-peptide–MHC II and capable of stimulating the same T cells in vitro. Thus, the mere presence of these apparently ‘immunogenic’ DCs in LNs was insufficient to generate a systemic response to self-antigen.

Infection of HEL transgenic mice with L. monocytogenes or anti-CD40 induces marked inflammation
To characterize the conditions that enabled the maintenance of peripheral tolerance despite the presence of apparently mature HEL peptide–MHC II-positive DCs, we determined the effect of further shifting the equilibrium toward inflammation. The steady-state function of DCs in lymphoid tissues is to induce tolerance in CD4+ and CD8+ T cells, by selectively targeting antigens to the DC population and observing that antigen-specific T cells became tolerant to challenge with that antigen and a strong adjuvant (17, 18, 33). Tolerance could be converted to immunity if the mice simultaneously were given anti-CD40 as a presumptive DC maturation stimulus. We asked if similar results would accrue in the case of HEL expressed as an endogenous self-antigen.

HEL-specific T cells were labeled with CFSE and injected into HEL transgenic or non-transgenic control mice. After 24 h, the mice were injected with L. monocytogenes or anti-CD40. Mice were sacrificed on day 4 or 8 and analyzed for T cell proliferation and splenic weight. Because the T cells injected into HEL transgenic animals had divided beyond the limits of detection of CFSE (Supplementary Figure 1, available at International Immunology Online), we instead monitored TCR down-regulation as a measure of T cell activation, labeling T cells removed from the adoptively transferred mice with CD4 and Vß8. As shown in Fig. 5, T cells gated on CD4 and analyzed for Vß8 expression fell into three general populations, that falling farthest to the right being the least activated.



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  		Fig. 5. In mice injected with HEL-specific T cells, and subsequently infected with Listeria monocytogenes or injected with anti-CD40, self-antigen-specific T cells are activated. Naive 3A9 TCR transgenic T cells were harvested from female mice as described. T cells were labeled with CFSE. Cells (5 x 106) were injected i.v. into 12-week-old female HEL transgenic or non-transgenic mice. Twenty-four hours after adoptive transfer, mice were injected i.p. with 103 L. monocytogenes, or 90 µg of anti-CD40. On day 4 and day 8 post-adoptive transfer, T cells were harvested from spleen and LNs. LN cells were pooled from each group, gated on CD4 and analyzed for Vß8 to detect TCR down-regulation. Shown is TCR expression on T cells harvested from (A) HEL transgenic and (B) non-transgenic mice. (C) 3A9 T cells were transferred into mice, and mice were injected with L. monocytogenes or anti-CD40, as described. T cells were isolated from these mice and incubated with DCs + 1 mg ml–1 HEL + LPS or no HEL.

 
It was clear that T cells isolated from mHEL mice had undergone extensive activation and therefore down-regulated their TCR expression, as shown by the shift of fluorescence to the left (Fig. 5A). T cells injected into non-HEL transgenic mice did not down-regulate their TCR even upon injection with L. monocytogenes (Fig. 5B). Thus, the TCR down-regulation was self-antigen specific and not a result of L. monocytogenes-specific T cells responding to bacterial infection. T cells from mHEL mice injected with anti-CD40 also exhibited an extensive activation in comparison to the mice injected with 3A9 only and non-HEL transgenic mice injected with anti-CD40.

As shown previously, T cells isolated from uninfected HEL transgenic mice were initially able to divide, and then became refractory to division when they were removed from the mice (Fig. 4B), and did not make IL-2 in response to antigen. When the HEL transgenic mice were infected with L. monocytogenes, the HEL-specific T cells divided extensively along with the L. monocytogenes-specific T cells. These T cells were not refractory to subsequent stimulation and continued to make IL-2 in response to antigen when they were removed from the HEL transgenic mice (Fig. 5C). This suggests that the nature of the signal that caused the T cells to divide is qualitatively different in the two cases. The 3A9 TCR transgenic T cells that had been transferred into non-HEL transgenic mice did not make significant IL-2 in response to antigen. Most likely, these T cells did not expand, whereas the T cells specific for L. monocytogenes did expand; therefore, fewer of the T cells placed into the ELISA were HEL specific.

The weights of the spleens were also consistent with a strong T cell expansion in the HEL transgenic mice, as the spleens isolated from mHEL mice that had been adoptively transferred were significantly larger than spleens from non-transgenic mice, even those infected with L. monoctyogenes or injected with anti-CD40 (data not shown). The health of the mHEL transgenic mice injected with L. monocytogenes or anti-CD40 provided the final clue that the T cells specific for an endogenous self-antigen had become activated. By day 7, the infected mHEL mice were lethargic, and often huddled in a corner of their cages. Their fur was matted and clumpy. This was not solely a result of infection because the mice injected with anti-CD40 had similar characteristics. Furthermore, the infected non-HEL transgenic mice did not appear to be as ill as the transgenic mice. In one experiment of eight HEL transgenic mice infected, one died at 3 weeks, two developed severe skin lesions and were sacrificed at 5 weeks and the rest of the mice survived for 9 months or more with no noticeable pathology. Because the HEL antigen is not localized to one organ as in characterized mouse models of autoimmune disease, a defined autoimmune pathology was not consistently observed. However, in one experiment, an HEL transgenic mouse (with adoptively transferred HEL-specific T cells) also developed paralysis following infection with Listeria, characteristic of that seen in experimental autoimmune encephalitis. This suggested that the illness was at least exacerbated by an autoimmune reaction rather than by infection alone.

Taken together, these observations suggest that immunogenic presentation of endogenous self-antigen required more than the simple presence of CD86+, HEL peptide–MHC II-expressing DCs in LNs, but a general shift of immunological status toward the inflammatory state.

DCs from anti-CD40-injected mice exhibit both ‘immunogenic’ and tolerogenic phenotypes
In order to examine the consequences of an inflammatory stimulus in vivo, and to investigate the presence of a candidate tolerogenic DC population, we studied LN DCs from mice that were injected with PBS or anti-CD40. As shown in Fig. 6, LN-derived DCs (Fig. 6B) are more heterogenous in regard to CD86 expression than bone marrow-derived DCs (Fig. 6A). Significantly, in the LN there is a population of DCs that is MHC class II high and CD86 low (Fig. 6B) that is absent in in vitro-derived bone marrow cultures (Fig. 6A). This population is found in both unstimulated and anti-CD40-injected mice. Because this population has high levels of MHC class II, but lacks co-stimulatory molecules, it is tempting to speculate that this population may be responsible for tolerance induction, although we have no direct evidence that this is the case.



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  		Fig. 6. In contrast to bone marrow-derived DCs, a sub-population of LN DCs express high levels of MHC-II molecules and low levels of CD86. (A) Bone marrow-derived DCs were derived as described above. Day 5 DCs were pulsed with anti-CD40, and 1 day later cells were analyzed for MHC class II and CD86 by FACS. (B) C3H/HeJ mice were injected i.p. with PBS or anti-CD40 (90 µg). Two days later, LN cells were harvested and enriched for CD11c+ cells as described in Fig. 3. These cells were stained for I-Ek and CD86 with antibodies 14-4-4 and GL1, respectively.

 
As shown in Fig. 6(B), as many as 33% of the DCs express abundant MHC II with low levels of CD86, regardless of whether mice have received an inflammatory stimulus. Comparing numbers of these possibly ‘tolerogenic DCs’ to the co-stimulator-high ‘immunogenic’ population, it becomes evident that in the presence of inflammation, there is a decrease in the ratio of tolerogenic to immunogenic DCs in the LN: in the steady state, tolerogenic outnumber immunogenic DCs by a 1.6:1 ratio, while after anti-CD40 injection, the ratio becomes 0.3:1.

In vitro, inflammation is known to up-regulate MHC class II and CD86. The same is true in vivo: inflammation, in this case anti-CD40, causes a significant increase in MHC class II (Supplementary Figure 2A, available at International Immunology Online, upper panels) and CD86 (Supplementary Figure 2B, available at International Immunology Online). The population of MHC class II-high, CD86-low cells remains, albeit at a lower percentage of DCs (Supplementary Figure 2A, available at International Immunology Online, lower panels). Together, these data suggest that tolerogenic DCs exist in both normal and anti-CD40-injected mice, but that they are outcompeted by an increased fraction of ‘inflammatory DCs’ in mice that are undergoing an inflammatory response.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary material
 References
 
Autoimmunity is often attributed to failed thymic tolerance, resulting in an autoreactive T cell repertoire. Although most autoreactive T cells are deleted in the thymus, reducing both the frequency and affinity of autoreactive T cells in the periphery, escape from negative selection is actually fairly common. Potentially autoreactive cells are readily detected in the periphery of both humans and mice (34–36). Thus, the shift to autoreactivity likely requires the autoreactive T cell and a relatively high-frequency auto-antigen in the periphery.

Peripheral tolerance is classically thought to be induced when an antigen-specific T cell encounters an APC (i.e. a DC) that expresses its cognate peptide–MHC complex in the absence of co-stimulatory molecules such as CD80 and CD86 (1, 2). Under such circumstances, T cells can be stimulated to divide several times, but eventually cease dividing or apoptose. Antigen presentation in the presence of co-stimulatory molecules, on the other hand, more often leads to T cell activation and sustained proliferation.

When considering the role of DCs in peripheral tolerance, it therefore seems reasonable to suggest that immature DCs, which express very low levels of co-stimulatory molecules, would thus be tolerogenic while mature DCs would be immunogenic. Indeed, in vivo, the balance between tolerance and immunity can be shifted by injecting stimuli of DC maturation together with DC-targeted antigen (18). One problem with this simple paradigm is that, at least in vitro, immature DCs often express low levels of peptide–MHC (1, 4). This appears to be true for many exogenous antigens presented either on MHC II (8, 37) or on MHC I (11). Conceivably, the low amounts of peptide–MHC that are generated by immature DCs are sufficient to provide a tolerogenic stimulus. Even if this were the case, the fact that maturation markedly enhances the number, and possibly the range, of peptide–MHC complexes formed creates the possibility that self-peptides may be presented by immunostimulatory mature DCs that were not effectively presented by tolerogenic immature DCs. One classically assumed mechanism that might tend to counteract this potential problem is peptide competition. When a DC is induced to mature, it has long been assumed that the presence of excess foreign antigen (e.g. bacteria, microbes) would generate peptides that might compete with endogenous or self-peptides for binding to a limited number of MHC molecules. We present data demonstrating that this mechanism cannot solely be responsible for preventing autoimmunity.

Although much attention has been paid recently to the immunogenic versus tolerogenic presentation of exogenous antigens on MHC I and MHC II by DCs (18), little is known about the presentation of endogenous self-antigens. We have explored this here and in the process addressed a few of the key assumptions associated with the current concepts of how DCs mediate tolerance versus immunity.

While the basic rules established in vitro for the presentation of exogenous antigens by DCs also apply to the presentation of transgenic HEL, it is also clear that immature DCs nevertheless produce sufficient HEL peptide–MHC II to generate a T cell response, at least when artificial co-stimulation is provided by anti-CD28. Indeed, the presentation of endogenous HEL by immature DCs appeared to be more efficient than exogenous HEL, suggesting that induction of tolerance to endogenous antigens may be more efficiently accomplished.

However, we also found that the production of endogenous self-HEL peptide–MHC II greatly increased upon DC maturation and that its presentation was not significantly decreased by the presence of foreign antigens. Thus, mature DCs have the ability to present self in the presence of abundant co-stimulatory molecule expression, a situation that might break tolerance induced by immature DCs, unless the deletion of autoreactive T cells or induction of regulatory T cells had been completely effective.

Under steady-state conditions in vivo, it was apparent that the HEL transgenic mice were capable of inducing tolerance to self-HEL, as the adoptive transfer of antigen-specific naive T cells resulted in only their transient proliferation. We did not evaluate the potential contribution of various APC populations to this response, but instead focused on CD11c+ DCs. As expected from work with antigens exogenous to the presenting DC, delivery of a DC maturation signal changed the picture rather dramatically, resulting instead in dramatic activation and proliferation of the transferred autoreactive T cells. While this result cannot be categorized as indicating the breaking of tolerance (the mice were not allowed to ‘tolerize’ the T cells prior to the injection of anti-CD40 or L. monocytogenes), the finding did confirm for an endogenous self-antigen that overall inflammatory status plays a critical role in determining whether immunity or tolerance ensues.

A surprise from these experiments emerged from an analysis of the DCs present in LNs under the two conditions. Even in the absence of an inflammatory stimulus, under conditions leading to apparent tolerance of adoptively transferred T cells, a large population of DCs isolated from nodes were phenotypically mature, expressing high levels of CD86. While CD86-positive DCs have been identified previously in lymphoid tissues (33), they had been thought not to contribute to the induction of tolerance versus immunity since, as mature DCs, they were unlikely to be efficient at internalizing exogenously administered antigen. In our experiments, however, HEL peptide–MHC II was clearly expressed on the surfaces of these CD86+ DCs both by staining with a mAb (data not shown) and by stimulation of naive 3A9 T cells in the absence of anti-CD28 in vitro.

Despite the presence of immunostimulatory, phenotypically mature DCs in the LNs of resting HEL transgenic mice, the adoptive transfer experiments clearly indicate that tolerance nevertheless develops. This paradox has several possible explanations. Conceivably, there is a small population of phenotypically immature DCs which detects the autoreactive T cells before they reach the peptide–MHC II-bearing mature DCs. These cells could be the MHC II-high, CD86-low cells detected in the LN. Such a population might contribute to the production of regulatory T cells that also help dampen the autoreactive response. Perhaps a more attractive alternative, however, would be to re-evaluate exactly what is meant or understood by DC maturation. Classically, a DC expressing high surface MHC II, peptide–MHC and co-stimulatory molecules is defined as having a mature ‘phenotype.’ However, not all such DCs may be functionally mature with respect to T cell outcome. It is possible that the simple paradigm of T cell encounter with or without co-stimulatory molecules as being the major determinant of tolerance versus immunity is itself not entirely accurate.

A functionally mature, immunostimulatory DC might have other phenotypic characteristics apart from the formation of peptide–MHC or the expression of co-stimulatory molecules. Such characteristics may include particular patterns of cytokine secretion (e.g. IL-6, IL-12, IFN-{alpha}). If so, there might be intermediate steps on the pathway of DC maturation, one of which would allow a phenotype optimized for the induction of tolerance. Such a phenotype may be characterized by an enhanced capacity for antigen processing and a partial expression of co-stimulatory molecules such as CD86. This intermediate step might be reached in the absence of a frank inflammatory stimulus, perhaps induced by the events associated with the constitutive migration of DCs from the periphery to the LN. When a true inflammatory signal is detected, however, migrating or LN resident DCs might complete the maturation pathway thus resulting in a highly immunostimulatory cell. Such a situation might also explain why tolerance is so often broken in clinical conditions, such as chronic viral infection, that may shift the maturation pathway in the direction of immunostimulation.


   Supplementary material
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material is available at International Immunology Online.


   Acknowledgements
 
We would like to acknowledge Ralph Steinman for his valuable input during scientific discussions. We would like to thank Christophe Viret for his technical expertise with adoptive transfers. We would also like to thank Rose Rullan and Rachael Manna for their excellent technical assistance. We are grateful to the Ludwig Institute for Cancer Research and the National Institutes of Health for their support.


   Abbreviations
 
APC   antigen-presenting cell
CFSE   5(6)-carboxyfluorescein diacetate N-succinimidyl ester
DC   Dendritic cell
HEL   hen egg lysozyme
i.p.   intraperitoneally
i.v.   intravenously
LB   Luria broth
LN   lymph node
MCC   moth cytochrome C
mHEL   Membrane hen egg lysozyme
PCC   pigeon cytochrome C
sHEL   soluble hen egg lysozyme
TPB   tryptose phosphate broth

   Notes
 
Transmitting editor: K. Inaba

* These authors contributed equally to this work. Back

Received 18 October 2004, accepted 31 October 2005.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary material
 References
 

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D. M. Turley and S. D. Miller
Peripheral Tolerance Induction Using Ethylenecarbodiimide-Fixed APCs Uses both Direct and Indirect Mechanisms of Antigen Presentation for Prevention of Experimental Autoimmune Encephalomyelitis
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