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The Journal of Immunology, 2008, 181, 3212 -3220
Copyright © 2008 by The American Association of Immunologists, Inc.
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The Relative Efficiency of Acquisition of MHC:Peptide Complexes and Cross-Presentation Depends on Dendritic Cell Type1

Lesley Ann Smyth*, Nicola Harker{dagger}, Wayne Turnbull{ddagger}, Haytham El-Doueik{ddagger}, Linda Klavinskis{ddagger}, Dimitris Kioussis{dagger}, Giovanna Lombardi* and Robert Lechler2,*

* Department of Nephrology and Transplantation, King’s College London, School of Medicine, Guy’s Hospital, {dagger} National Institute for Medical Research, Division of Molecular Immunology, {ddagger} Peter Gorer Department of Immunobiology, King’s College London, School of Medicine, Guy’s Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intercellular exchange of MHC molecules has been reported between many cells, including professional and nonprofessional APCs. This phenomenon may contribute to T cell immunity to pathogens. In this study, we addressed whether the transfer of MHC class I:peptide complexes between cells plays a role in T cell responses and compare this to conventional cross-presentation. We observed that dsRNA-matured bone marrow-derived dendritic cells (BMDCs) acquired peptide:MHC complexes from other BMDCs either pulsed with OVA257–264 peptide, soluble OVA, or infected with a recombinant adenovirus expressing OVA. In addition, BMDCs were capable of acquiring MHC:peptide complexes from epithelial cells. Spleen-derived CD8{alpha}+ and CD8{alpha}– dendritic cells (DCs) also acquired MHC:peptide complexes from BMDCs pulsed with OVA257–264 peptide. However, the efficiency of acquisition by these ex vivo derived DCs is much lower than acquisition by BMDC. In all cases, the acquired MHC:peptide complexes were functional in that they induced Ag-specific CD8+ T cell proliferation. The efficiency of MHC transfer was compared with cross-presentation for splenic CD8{alpha}+ and CD8{alpha}– as well as BMDCs. CD8{alpha}+ DCs were more efficient at inducing T cell proliferation when they acquired Ag via cross-presentation, the opposite was observed for BMDCs and splenic CD8{alpha}– DCs. We conclude from these observations that the relative efficiency of MHC transfer vs cross-presentation differs markedly between different DC subsets.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Within the context of infection, the activation of naive CD8+ T cells occurs either by direct priming or cross-presentation (1, 2, 3, 4, 5). Direct priming occurs when CD8+ T cells recognize their cognate MHC:peptide complexes on the surface of the cell that synthesized the Ag, such as a virally infected APC. Priming of CD8+ T cells by CD8{alpha}+ dendritic cells (DCs)3 presenting virally derived peptides has been demonstrated (6, 7). In addition CD8{alpha}– DCs, such as Langerhans cells and dermal derived DCs, can also prime CD8+ T cells (6). However, for viruses that do not directly infect or replicate poorly within the APC naive CD8+ T cells can be primed by APCs that have acquired, processed, and presented exogenous Ags through the class I pathway (8, 9, 10, 11, 12). This phenomenon has been referred to as cross-priming (4). In fact DCs, predominantly the CD8{alpha}+ subset, appear to be the main cell type capable of acquiring and presenting Ag in this manner (1, 13, 14). CD8{alpha}+ DCs have been shown to cross-present i.v. introduced soluble Ag, that gains access to the spleen, as well as cell-associated Ags (13, 14, 15). It has been reported that lymph node resident CD8{alpha}+ DCs can capture cellular Ags from trafficking CD8{alpha}– DCs and present them to naive CD8+ T cells (16). Recently, it has been shown that only a subset of CD8{alpha}+ DCs are efficient at cross-presentation and that these DCs express abundant IL-12 and TLR3 (17). Bone marrow-derived DCs (BMDCs) are also capable of cross-presenting Ag (18, 19).

However a third mechanism may also exist. We, and others, have shown that class I MHC molecules can be acquired by both immature and mature DCs in vitro and in vivo (20, 21, 22, 23). Importantly, we also reported that allogeneic MHC class I molecules acquired in vitro and in vivo induce proliferation of allospecific CD8+ T cells. We named this the "semidirect" pathway of allorecognition (20).

It is possible that this intercellular transfer of MHC:peptide complexes may play a part in immunity against infectious agents such as viruses. As for direct priming and cross-presentation, DCs appear to be pivotal in this process and as both immature and mature DCs are capable of acquiring MHC molecules it is possible that this phenomenon can take place in both the peripheral and lymphoid tissue. DCs trafficking through virally infected tissue may well use this pathway to acquire, and subsequently present, viral peptide:MHC complexes in the lymph nodes to T cells. It could also be envisaged that DCs pass MHC:peptide complexes on to lymph node resident CD8{alpha}+ and CD8{alpha}– DCs, which, in turn, induce T cell priming.

The purpose of this study was to compare the efficiency of MHC:peptide complex acquisition with cross-presentation in different DC subtypes. If the efficiency of these two mechanisms of Ag presentation are comparable, this finding would increase the likelihood that MHC:peptide complex acquisition is of biological significance in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cell lines, and Abs

BALB/c (H-2d) and C57BL/6 (H-2b) mice, 6–10 wk of age were purchased from Harlan Olac. OT-1 mice were purchased from The Jackson Laboratory. Mice were kept under sterile conditions. Mouse handling and experimental procedures were conducted in accordance with national and institutional guidelines for animal care and use. The H-2b-expressing murine epithelial cell line (YO1) was a gift from D. Kioussis, (National Institute for Medical Research, London, U.K.) (24).

All the mAbs used, unless stated otherwise, were purchased from BD Biosciences.

DC cultures

Mouse BMDCs were generated as follows. Briefly, bone marrow was flushed from femurs, passed through a 70-µM nylon cell strainer (BD Biosciences), and RBCs were lysed using ACK buffer. Cells were then incubated with a mixture of rat mAbs to B220 (culture supernatants from RA3-6B2 hybridoma), MHC class II (culture supernatants from MS/114.52 hybridoma), anti-CD8 (culture supernatant from 53 to 6.72 hybridoma) and anti-CD4 (culture supernatant from YTS191 hybridoma) followed by an incubation period with sheep anti-rat-IgG-coated Dynabeads (Dynal Biotech). The bead-mAb bound cells were selected using a magnet. Remaining cells were cultured at 1 x 106/ml in RPMI 1640 (Life Technologies) containing 10% FCS, 50 µM 2-ME, 100 IU/ml penicillin, 100 mg of streptomycin, 2 mM L-glutamine, and 1% HEPES and 6 ng/ml mouse recombinant GM-CSF (DC medium). On day 2 and 4 of culture, floating cells were gently removed and fresh mouse recombinant GM-CSF was added. On day 6 of culture, BMDCs were either left untreated or induced to mature using 10 µg/ml polyinosinic-polycytidylic acid (Amersham Biosciences). After overnight culture cells were harvested. For purity analysis of BMDC, cells were first incubated with an anti-CD16/CD32 (anti-FcR{gamma}III/FcR{gamma}II, clone 2.4G2) mAb for 10 min and subsequently stained with PE-conjugated anti-CD11c mAb (clone HL3) or isotype matched Abs. The DCs purity was consistently between 90 and 95%.

Spleens from mice were disaggregated using collagenase and DNase. CD8{alpha}+ and CD8{alpha}– splenic DCs were purified using a CD8{alpha}+ DC purity kit from Miltenyi Biotec following the manufacturers instructions. CD8{alpha}– DCs were further purified via flow cytometry. For analysis of purity, cells were first incubated with an anti-CD16/CD32 (anti-FcR{gamma}III/FcR{gamma}II, clone 2.4G2) mAb for 10 min and subsequently stained with PE-conjugated anti-CD11c mAb (clone HL3) and FITC conjugated anti-CD8{alpha} (clone 53–6.72) or isotype matched control Abs. The purity of CD8{alpha}+ and CD8{alpha}– DCs was consistently >90%.

Ag and electroporation

OVA257–264 peptide was used at 10 µg/ml. Soluble OVA protein (OVA) was purchased from Sigma-Aldrich and used at a concentration of 4 mg/ml. Soluble OVA was introduced into DCs via electroporation as previously described (25). Preparation of recombinant adenovirus (rAd) expressing cDNA for soluble OVA or GFP has been described elsewhere (26). In this study, day 6 BMDCs were infected with 3000 virus particles per cell. This dose of virus has been previously used to transduce murine BMDCs both by our group and others (26, 27).

MHC class I transfer experiments

MHC donor DCs (H-2b) were re-suspended at 107 cells/ml in a 2 µM solution of CFSE (Molecular Probes) in PBS and incubated for 10 min in the dark. At the end of the incubation period, cells were washed twice with cold PBS containing 10% FCS. A total of 5 x 105 cells CFSE-labeled MHC "donor" BMDCs (H-2b) were then cocultured with equal numbers of unlabelled MHC "recipient" BMDCs, CD8{alpha}+ or CD8{alpha}– splenic DCs (H-2d) in 1 ml of DC medium in a 24-well plate for 20 h. The next day cells were either stained with H-2b to check for MHC transfer or sorted using a MoFlo high-speed cell sorter (DakoCytomation) machine on the biases of their CFSE label. For optimal purity CFSE-negative cells were sorted twice and the purity was always >99% (data not shown).

MHC transfer and flow cytometry

For analysis of MHC transfer, cells were firstly incubated with the anti-CD16/CD32 (2.4G2 hybridoma) Abs and then stained with specific PE-conjugated H-2b or equivalent isoptype control Abs. Analyses were performed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest acquisition and analysis software on cells gated for homogenous forward scatter and side scatter characteristics. The 25-D1.16 Ab was a gift from R. Germain (National Institutes of Health, Bethesda, MD) (28).

Preparation of responder T cells

Responder T cells were purified from splenocytes from OT-1 mice. RBCs depleted leukocytes were incubated with a mixture of rat mAbs to B220 (RA3-6B2 hybridoma), MHC class II (MS/114.52 hybridoma), anti-CD16/CD32 (2.4G2 hybridoma), and anti-CD4 (YTS191 hybridoma) followed by an incubation period with sheep anti-rat-IgG-coated Dynabeads (Dynal Biotech). The bead- or mAb-bound cells were selected using a magnet and purified CD8+ T cell populations were recovered from the fluid phase. The purity of responder T cells was assessed using PE-conjugated anti-CD8 Abs (clone 53-6.7). The purity of T cells was consistently between 90 and 95%.

T cell proliferation and CTLL assays

A total of 2.5 x 104 purified CD8+ T cells were stimulated with 2.5 x 104 sorted DCs in triplicate wells of a 96-well plate. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture. Results are shown as mean cpm ± SD of triplicate determinations. To measure IL-2 production, 5 x 103 CTLL were incubated with 50 µl of supernatant, taken from the described cultures, for 16 h and proliferation measured by [3H]thymidine incorporation after 2 days in culture. Results are shown as mean cpm ± SD of triplicate determinations.

Cross-presentation assays

Cross-presentation assays were set up as previously described (29) but with slight modifications. Briefly, 2.5 x 105 CD8{alpha}2+ splenic DCs, CD8{alpha}– splenic DCs or BMDC, expressing H-2b were plated in 96-well flat-bottom plate in DC medium with 2.5 x 105 T cells derived from OT-1 mice and 2.5 x 105 H-2d expressing BMDCs that had been loaded with soluble OVA and 10 µg/ml polyinosinic-polycytidylic acid via electroporation. T cell proliferation was assessed after 3 days using [3H] incorporation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
dsRNA-matured BMDCs can acquire intact MHC class I:peptide complexes from other mature BMDCs

DCs can acquire preformed peptide:MHC complexes from other cells through direct contact or through the uptake of exosomes (30) both in vivo and in vitro as previously demonstrated by our group and others in the context of allorecognition (20, 30). It is possible that acquisition of Ag via MHC transfer may also be used to active CD8+ T cells during infection.

We have previously shown that MHC:peptide complexes can be donated and acquired by LPS-mature BMDCs (20). During a viral infection, however, DCs may be matured by viral dsRNA through the recognition of TLR3 (31). To assess whether dsRNA-matured BMDCs can donate and acquire MHC class 1 molecules we analyzed MHC acquisition in vitro using a coculture system whereby dsRNA-matured BMDCs from two different strains of mice (C57BL/6 (B6) and BALB/c) were mixed. To distinguish each cell population MHC donor BMDC derived from B6 mice (H-2b) were CFSE-labeled, before being cocultured with the recipient BALB/c BMDCs (H-2d). Labeling of the donor cells and subsequent analysis of CFSE-negative recipient cells excludes the possibility of mistakenly analyzing doublets comprising cells from the two input populations. Following 20 h of coculture, staining with an anti-H-2Kb Ab showed that the whole population of BALB/c BMDCs shifted to the right on a single parameter flow cytometric histogram (Fig. 1A). This observation suggests that MHC transfer could occur between two dsRNA-matured DCs. To confirm that the acquired allogeneic MHC molecules were fully functional, MHC donor DCs were loaded with OVA257–264 peptide, before being CFSE-labeled and cocultured with MHC recipient DCs. After 20 h, CFSE-negative (BALB/c) cells were selected by flow cytometric cell sorting and were used to stimulate OVA-specific transgenic CD8+ T cells. We observed that MHC recipient BMDCs were able to induce both proliferation and IL-2 production, as measured by CTLL proliferation, of TCR-transgenic CD8+ T cells that recognize OVA257–264 peptide in the context of H-2Kb. T cell responses compared favorably to that induced by peptide-pulsed B6 BMDC (Fig. 1B) and was higher than that of BALB/c DCs pulsed with OVA257–264 peptide (Fig. 1C). In contrast, MHC recipient DCs cocultured with unpulsed donor DCs failed to stimulate T cell proliferation or IL-2 production (Fig. 1B).


Figure 1
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  		FIGURE 1. The dsRNA-matured BMDCs can acquire MHC class 1 molecules. A, BALB/c BMDCs (H-2d) were cocultured with CFSE-labeled B6 BMDCs (H-2b). Both sets of BMDCs had been previously matured with dsRNA. After 20 h of coculture, cells were stained with PE-conjugated anti-H-2Kb mAb (gray filled histogram) or control isotype Ab (filled histograms). Analysis was done on CFSE-negative cells. B, An equal number of dsRNA-matured BALB/c BMDCs were mixed with CFSE-labeled B6 BMDCs that had previously been pulsed for 4 h with OVA257–264 peptide. BALB/c BMDCs cocultured with unpulsed CFSE-labeled B6 BMDCs served as controls. After 20 h of coculture cells, CFSE-negative BALB/c BMDCs were separated from CFSE-expressing B6 BMDCs via flow cytometric sorting. A total 2.5 x 104 DCs were used to stimulate 2.5 x 104 OVA-specific CD8+ T cells and T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture (top). Production of IL-2 was measured by incubating 5 x 103 CTLL cells with 50 µl of supernatant taken from these cultures. Proliferation was measured after 3 days in culture (bottom). Results are represented as the mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*). C, Experimental design as described in B. CFSE-negative BALB/c BMDCs isolated following coculture were highlighted (*) and compared with BALB/c BMDCs pulsed with OVA257–264 peptide. Proliferation was measured after 3 days in culture. Results are represented as the mean cpm ± SD of triplicate determinants.

 
BMDCs can acquire MHC:peptide complexes from mature BMDCs even when the Ag levels are limited

To assess whether MHC transfer could occur when Ag concentration is limited we loaded dsRNA-matured MHC class I donor BMDCs with soluble OVA via electroporation, as previously described (25). This route of Ag presentation requires proteasome activity and export of MHC class I molecules from the endoplasmic reticulum (25). Reis e Sousa and Germain (32) have detected peptide contamination in different batches of OVA. Two approaches were used to test our OVA preparations for peptide contamination. First, we fixed B6 BMDC with glutaraldehyde (25), and incubated them with either OVA257–264 peptide or soluble OVA before adding to OVA-specific CD8+ T cells. Fixed BMDCs were capable of presenting OVA257–264 and inducing T cell proliferation (Fig. 2A). In contrast, fixed BMDCs were incapable of processing soluble OVA protein and did not induce any T cell proliferation above that induced by unpulsed B6 BMDC (Fig. 2A) indicating that no functionally significant concentrations of peptide were present in the soluble OVA preparations used in this experiment. Second, we dialyzed soluble OVA for 36 h at 4°C with several changes of PBS to remove any small peptides (less than 12 kDa) using a method described by Stoitzner et al. (33) and compared the OVA-specific CD8+ T cell response to B6 BMDC loaded with either dialyzed or nondialyzed soluble OVA. The T cell response to OVA before and after dialysis was comparable (Fig. 2B). This observation was in agreement with the data of the aforementioned study. Both these observations suggested that significant concentrations of peptide were not present in our soluble OVA preparations.


Figure 2
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  		FIGURE 2. Soluble OVA did not contain detectable levels of contaminating peptides. A, B6 BMDCs were either fixed with 0.0008% glutaraldehyde or were left untreated, and incubated with soluble OVA or OVA257–264 peptide for 2 h at 37°C. Controls received no Ag. A total of 2.5 x 104 DCs were incubated with an equal number of OVA-specific CD8+ T cells and proliferation was measured by [3H]thymidine incorporation after 3 days in culture. B, B6 BMDCs were incubated with either undialyzed or dialyzed soluble OVA. Controls received no Ag. A total of 2.5 x 104 DCs were then incubated with an increasing number of OVA-specific CD8+ T cells. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture.

 
To test the amount of MHC:peptide complexes available following peptide pulsing or electroporation we incubated peptide pulsed and soluble OVA electroporated BMDCs derived from B6 mice with an Ab that recognizes OVA257–264/H-2Kb complexes (25-D1.16). We observed that the level of peptide:MHC complexes on BMDC derived from B6 mice following electroporation with soluble OVA was substantially lower than that following pulsing cells with OVA257–264 peptide as measured after 3 and 24 h (Fig. 3A).


Figure 3
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  		FIGURE 3. Mature BMDCs can acquire MHC:peptide complexes even when the Ag levels are limited. A, Mature B6 BMDCs were either pulsed with OVA257–264 peptide (solid gray histogram) or electroporated with soluble OVA (dotted histogram) as described in Materials and Methods. After 3 (top) and 24 (bottom) h, DCs were stained with 25-D1.16 mAb followed by anti-mouse IgG-biotin and streptavidin PE. Controls DCs were unpulsed cells (solid black histogram). B, An equal number of dsRNA-matured BALB/c BMDCs were mixed with CFSE-labeled mature B6 BMDCs in which soluble OVA had been introduced via electroporation. After 20 h of coculture cells were separated via flow cytometric sorting. A total of 2.5 x 104 DCs were used to stimulate an equivalent number of OVA-specific CD8+ T cells. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture (top). Results are represented as the mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*). In a separate experiment, CFSE-negative BALB/c BMDCs isolated following coculture, highlighted (*), were compared with BALB/c BMDCs either electroporated or not with soluble OVA. Proliferation was measured after 3 days in culture (bottom). Results are represented as the mean cpm ± SD of triplicate determinants.

 
Following electroporation MHC donor B6 BMDCs were CFSE-labeled and cocultured with recipient BALB/c BMDCs for 20 h before being sorted by flow cytometry on the basis of their CFSE positivity. Both DC populations had been matured with dsRNA. CFSE-positive (H-2b) and CFSE-negative (H-2d) cells were then used to stimulate OVA-specific transgenic T cells. We observed that recipient BMDCs stimulated an Ag-specific T cell response following coculture with MHC donor DCs pulsed with soluble Ag (Fig. 3B, top) and this was higher than that of BALB/c DCs pulsed with soluble OVA (Fig. 3B, bottom). Recipient DCs cocultured with unpulsed donor DCs failed to stimulate a T cell response (Fig. 3B). However the overall T cell responses were lower than those observed following coculture with DCs pulsed with OVA257–264 peptide (Fig. 1B), presumably reflecting lower levels of MHC:peptide complexes present on donor DCs following electroporation (Fig. 3A). As expected, MHC donor BMDCs, loaded with soluble OVA protein induced both proliferation and IL-2 production (data not shown) by OVA-specific CD8+ T cells.

These results indicate that even under conditions in which the number of MHC:peptide complexes were limited mature DCs were capable of acquiring functional MHC:peptide complexes from dsRNA-matured DCs.

BMDCs can acquire MHC:OVA peptide complexes from other BMDCs infected with a rAd expressing OVA

To assess whether MHC:peptide complex transfer can occur between virally infected and uninfected DCs we incubated immature MHC donor DCs with a replication-deficient rAd carrying the cDNA for soluble OVA, before coculturing with mature recipient DCs. We have previously shown that immature BMDCs are susceptible to infection with rAd, leading to DC maturation (25). After 20 h of coculture, DCs were selected by flow cytometric sorting, as described, and used to stimulate OVA-specific CD8+ T cells. We observed that recipient BALB/c BMDCs stimulated an Ag-specific T cell response after being cocultured with MHC donor DCs infected with virus. No T cell response to recipient BMDCs cocultured with rAd-GFP infected B6 DCs was observed. As expected, from our previous findings, MHC donor BMDCs, derived from B6 (H-2b) mice infected with rAd-OVA induced proliferation of OVA-specific CD8+ T cells (Fig. 4).


Figure 4
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  		FIGURE 4. BMDCs acquire OVA peptide:MHC from DCs infected with a rAd expressing OVA. An equal number of dsRNA-matured BALB/c BMDCs were mixed with CFSE-labeled B6 BMDC infected with rAd-OVA, 3000 virus particles per cell. Controls were B6 BMDCs infected with rAd-GFP. After 20 h of coculture, cells were separated via flow cytometric sorting and 2.5 x 104 DCs were used to stimulate the equivalent number of OVA-specific CD8+ T cells. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture. Results are represented as mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*).

 
These findings suggest that MHC:peptide complex transfer maybe a mechanism whereby exogenous viral Ag is acquired by DCs.

Mature BMDCs can acquire peptide:MHC class I molecules presented by epithelial cells

Epithelial cells are susceptible to viral infection (34). In fact virally infected epithelial cells secrete many molecules that modulate the immune response, for example, upon viral infection by HIV, epithelial cells produce defensins (34, 35). These are chemotactic factors that recruit T cells and monocytes (34). In addition murine β-defensin recruits immature BMDC through CCR6 to the site of infection. This molecule also induces DC maturation through TLR4 (36). Additionally DCs recruited into inflamed epithelial tissues are responsible for priming CD8+ T cells through cross-presentation of Ag (37). These data suggest that interplay between DCs and epithelial cells during viral infection may occur. To investigate this suggestion, we cocultured a CFSE-labeled epithelial cell line expressing H-2b MHC molecules with H-2d expressing BMDCs, as described, and H-2b MHC acquisition was assessed by Ab staining and flow cytometric analysis. In these experiments BMDCs had been matured with dsRNA. Following overnight coculture, at least 35% of H-2b negative BMDCs expressed H-2Kb (Fig. 5A). Furthermore, H-2d expressing BMDCs cocultured either with H-2b expressing epithelial cells pulsed with OVA257–264 peptide or soluble OVA were capable of stimulating an Ag-specific CD8+ T cell response as measured by proliferation and IL-2 production. By contrast, H-2d expressing BMDCs cocultured with unpulsed H-2b expressing epithelial cells were unable to do so (Fig. 5B). We conclude that DCs trafficking through a virally infected site could acquire Ag in the form of preformed MHC:peptide class I complexes from virally infected epithelial cells and subsequently stimulate a T cell response.


Figure 5
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  		FIGURE 5. BMDCs can acquire peptide:MHC class I complexes from epithelial cells. A, dsRNA-matured BALB/c BMDCs (H-2d) were cocultured with a CFSE-labeled murine epithelial cell line expressing H-2b. After 20 h of coculture, cells were stained with PE-conjugated anti-H-2Kb mAb (gray line histogram) or control isotype Ab (filled histogram). Analysis of H-2Kb expression was done on CFSE-negative cells (BMDCs). The number shown represents the percentage of BALB/c DCs expressing the H-2Kb MHC molecules after coculture with CFSE-labeled H-2b expressing epithelial cells. B, An equal number of dsRNA-matured BALB/c BMDCs were mixed with CFSE-labeled epithelial cells that had previously been pulsed for 4 h with OVA257–264 peptide (top) or soluble OVA (bottom). BALB/c BMDCs cocultured with un-pulsed B6 BMDCs served as controls. After 20 h of coculture cells were separated via flow cytometric sorting and 2.5 x 104 DCs were used to stimulate equivalent numbers of OVA-specific CD8+ T cells. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture. Results are represented as the mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*).

 
Both CD8{alpha}+ and CD8{alpha}– DCs can acquire intact MHC:peptide complexes from BMDCs

As previously mentioned, resident CD8{alpha}+ DCs can acquire exogenous Ag from trafficking DCs and prime T cells (16, 38). Recently, Allen et al. (16) have shown that CD8+ T cell responses to HSV require cross-presentation of HSV proteins by CD8{alpha}+ DCs. It appears that HSV infected DCs migrate and "ferry" Ags to the lymph node and immediately transfer these Ags to CD8{alpha}+ DCs for cross-presentation. The source of exogenous Ag could be in the form of dying cells or cell debris, both of which would be phagocytosed and processed by the CD8{alpha}+ DCs. Alternatively, exogenous Ag could be acquired through transfer of preformed MHC:peptide complexes following cell to cell contact between the migratory DCs and the resident CD8{alpha}+ DC. To address this possibility, we cocultured splenic derived CD8{alpha}+ DCs (H-2d) with BMDCs (H-2b) pulsed with OVA257–264 peptide. In these experiments, BMDCs had been matured with dsRNA. As observed previously with BMDCs, H-2d expressing splenic CD8{alpha}+ DCs were capable of inducing an Ag-specific T cell response following coculture with H-2b expressing BMDCs pulsed with OVA257–264 peptide, albeit this response was markedly weaker than when BMDCs were used as recipient cells (Fig. 6A).


Figure 6
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  		FIGURE 6. CD8{alpha}+ and CD8{alpha}– splenic-derived DCs can acquire MHC class I molecules from BMDCs. An equal number of immature CD8{alpha}+ DCs (A) or CD8{alpha}– (B) DCs isolated from BALB/c mice were mixed with CFSE-labeled, dsRNA treated, B6 BMDCs that had previously been pulsed for 4 h with OVA257–264 peptide. CD8{alpha}+ or CD8{alpha}– splenic DCs cocultured with unpulsed B6 BMDCs served as controls. After 20 h, cells were separated via flow cytometric sorting and 2.5 x 104 DCs were used to stimulate an equivalent number of OVA-specific CD8+ T cells. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture (top). Production of IL-2 induced by CD8{alpha}+ DCs was measured by incubating 5 x 103 CTLL cells with 50 µl of supernatant taken from these cultures (A, bottom). Proliferation was measured after 3 days in culture. Results are represented as the mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*).

 
To address whether other splenic DCs were capable of acquiring MHC:peptide complexes we cocultured spleen-derived CD8{alpha}– DCs (H-2d) with BMDCs (H-2b) pulsed with OVA257–264 peptide. We observed that H-2d expressing splenic CD8{alpha}– DCs were capable of inducing an Ag-specific T cell response following coculture with H-2b expressing BMDCs pulsed with OVA257–264 (Fig. 6B). Therefore, we conclude that CD8{alpha}+ and CD8{alpha}– splenic DCs can acquire functional preformed MHC:peptide complexes from other DCs and activate CD8+ T cells.

MHC:peptide transfer is a more efficient route of Ag presentation than cross-priming for BMDC and CD8{alpha}– but not for CD8{alpha}+ splenic DCs

Except for viruses that infect DCs, cross-presentation of exogenous Ags through the uptake of soluble Ag, dying cells, or cell debris is thought to be a key mechanisms by which DCs prime T cells to viral Ags (8, 9, 10). Although CD8{alpha}+ DCs have been shown to be the main cell type responsible for cross-presentation, BMDCs have also been reported to be capable of cross-presenting cell-associated Ags (19). From our data presented, it is possible that transfer of intact MHC:peptide complexes is another mechanism by which exogenous Ags are acquired by both CD8{alpha}+ DCs and BMDCs. Unlike cross-presentation, Ag acquired through MHC transfer does not require further processing by the recipient DC to prime T cells. To compare the efficiency of MHC transfer and cross-presentation, we stimulated CD8+ T cells with DCs that had acquired MHC:peptide complexes via MHC transfer or with DCs that had acquired and processed cell surface Ag and measured T cell proliferation. B6 BMDC were loaded with soluble OVA, via electroporation, CFSE-labeled and cocultured with recipient BALB/c DCs for 20 h before flow cytometric cell sorting. Both CFSE-positive and CFSE-negative DCs were used to stimulate OVA-specific T cells. At the same time cross-presentation experiments were set up. B6 DCs were cocultured with an increasing number of BALB/c DCs loaded with soluble OVA and dsRNA, via electroporation, and CD8+ T cells. Controls were incubated with a similar number of BALB/c DCs that had been electroporated with dsRNA only. T cell activation in these cultures was dependent on B6 DCs (H-2b) acquiring and processing cell-associated OVA from OVA-loaded BALB/c DCs.

In agreement with our previous observations, MHC:peptide complexes were captured more efficiently by BMDCs compared with CD8{alpha}+ DCs as indicated by T cell proliferation (Figs. 7 and 8). Although the level of T cell activation induced by CD8{alpha}+ BALB/c splenic DCs cocultured with OVA loaded B6 BMDCs was low, it was significantly greater than following stimulation with BALB/c splenic DCs cocultured with unpulsed B6 BMDCs (data not shown).


Figure 7
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  		FIGURE 7. T cell activation by BMDCs is more efficient when the Ag is acquired via MHC transfer rather than cross-presentation. MHC transfer experiments were set up by mixing an equal number of dsRNA matured BALB/c BMDCs with CFSE-labeled B6 BMDCs that had previously been pulsed with soluble OVA via electroporation. BALB/c BMDCs cocultured with unpulsed B6 BMDCs served as controls. After 20 h of coculture, cells were separated on the bases of their CFSE positivity via flow cytometric sorting and 2.5 x 104 DCs were used to stimulate 2.5 x 104 OVA-specific CD8+ T cells. Cross-presentation was set as follows, 2.5 x 104 CD8+ T cells were cultured with an equal number of B6-derived BMDCs and either 2.5 x 104 (X1) or 1.25 x 105 (X5), 2.5 x 105 (X10), and 5 x 105 (X20) BALB/c derived BMDCs that had been pulsed with soluble OVA and dsRNA. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture. Results are represented as the mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*). Conditions in which cross-presentation had occurred are shown (^).

 

Figure 8
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  		FIGURE 8. T cell activation by CD8{alpha}+ splenic DCs is more efficient when the Ag is acquired via cross-presentation. MHC transfer experiments were set up by mixing equal numbers of CD8{alpha}+ splenic BALB/c BMDCs with CFSE-labeled B6 BMDCs that had previously been pulsed with soluble OVA and dsRNA via electroporation. BALB/c CD8{alpha}+ splenic DCs cocultured with unpulsed B6 BMDCs served as controls. After 20 h of coculture, cells were separated on the bases of their CFSE positivity via flow cytometric sorting and 2.5 x 104 DCs were used to stimulate 2.5 x 104 OVA-specific CD8+ T cells. Cross-presentation experiments were as follows, 2.5 x 104 CD8+ T cells were cultured with an equal number of CD8{alpha}+ splenic DCs derived from B6 mice and BMDCs pulsed with soluble OVA and dsRNA derived from BALB/c mice. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture. Results are represented as mean cpm ± SD of triplicate determinants. Conditions in which MHC transfer has occurred have been highlighted (*). Conditions in which cross-presentation had occurred are shown (^).

 
When comparing the efficiency of MHC transfer with cross-presentation we found that BMDCs, which had acquired exogenous Ag via MHC:peptide transfer, were capable of inducing stronger T cell responses than when they had acquired Ag via cross-presentation (Fig. 7). This was the case even when we increased the number of BALB/c DCs containing OVA and dsRNA (either 5, 10, or 20 fold) in the cross-presentation assays (Fig. 7). By contrast, CD8{alpha}+ DCs were capable of inducing a stronger T cell response when the Ag was acquired by cross-presentation rather than via MHC:peptide complex transfer (Fig. 8). This was also the case for FLT-3L generated BMDC CD8{alpha}+-like DCs (data not shown). No T cell stimulation occurred in the absence of B6 splenic CD8{alpha}+ or BMDCs (data not shown).

As these observations suggest a difference in the ability of DC subsets to induce T cell responses following acquisition of Ag either via cross-presentation or MHC transfer we decided to investigate the stimulatory capacities of another subset of spleen derived DCs, the CD8{alpha}– DCs. These cells have recently been described as being inefficient at cross-presentation of soluble Ag in vivo (39). We also observed this in vitro (Fig. 9). When comparing the efficiency of MHC transfer with cross-presentation in this DC subset we found that CD8{alpha}– DCs induced strong T cell responses when they had acquired Ag via MHC:peptide complex transfer rather than cross-presentation (Fig. 9).


Figure 9
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  		FIGURE 9. T cell activation by CD8{alpha}– DCs is more efficient when the Ag is acquired via MHC transfer rather than cross-presentation. MHC transfer experiments were set up as previously described. CFSE-labeled B6 BMDCs, loaded with soluble OVA, were cocultured with CD8{alpha}– splenic DCs isolated from BALB/c mice. CD8{alpha}– splenic DCs cocultured with unpulsed B6 BMDCs served as controls. After 20 h of coculture, cells were separated on the basis of their CFSE positivity via flow cytometric sorting and 2.5 x 104 CFSE-negative and CFSE-positive DCs were used to stimulate 2.5 x 104 OVA-specific CD8+ T cells. Conditions in which MHC transfer has occurred have been highlighted (*). Cross-presentation was set up using 2.5 x 104 CD8+ T cells cultured with an equal number of B6 CD8{alpha}– splenic DCs and either 2.5 x 104 (X1) or 1.25 x 105 (X5) BALB/c BMDCs that had been pulsed with soluble OVA and dsRNA. T cell proliferation was measured by [3H]thymidine incorporation after 3 days in culture. Results are represented as the mean cpm ± SD of triplicate determinants. Conditions in which cross-presentation had occurred are shown (*).

 
These experiments (Figs. 7–9) were performed in the presence of dsRNA. As TLR ligand has been shown either to down-regulate (40) or induce cross-presentation (18, 31) we tested whether this TLR ligand affected the ability of each of the aforementioned DC subtypes to cross-present Ag. We observed that in the absence of TLR signaling CD8{alpha}+ splenic DCs cross-presented OVA more efficiently, however, this was not the case for BMDCs and CD8{alpha}– DCs. No difference in cross-presentation was observed in the absence or presence of TLR signal with these DCs (data not shown).

We conclude from these observations that the relative efficiency of MHC transfer vs cross-presentation differs markedly between different DC subsets.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
During infection, naive T cells are primed either by virus-infected DCs presenting viral Ags or by uninfected DCs that have acquired viral Ags through the uptake of infected/dying cells or cell debris. In this study, we have investigated a third pathway. T cells can be primed by uninfected DCs that have acquired Ag in the form of preformed MHC:peptide complexes, from infected cells. Transfer of MHC:peptide complexes between cells has recently been termed ‘cross-dressing’ (21, 22, 41). Although transfer of molecules has been shown in vitro, whether this phenomenon occurs in vivo, as well as its biological relevance is still a subject of much debate and continuing investigation, as discussed later (30). The transfer of MHC:peptide complexes between cells may enhance CD8+ T cell priming to Ags that are not efficiently presented, as would be the case with tissue-specific viruses that do not infect DCs or if the virus impairs the immune function of the DC it has infected. Although cross-presentation may be important for immunity under these circumstances not all T cell epitopes are efficiently cross-presented (8, 9, 10). A theoretical advantage of MHC:peptide acquisition from infected tissue cells is that it guarantees the faithful display of dominant peptides by trafficking DCs upon arrival in the lymph node. The complex series of steps required for Ag or apoptotic cell uptake, processing, and presentation do not offer the same guarantee.

Intercellular exchange of MHC molecules has been reported between many cells, including professional and nonprofessional APCs (30, 42). However, MHC acquisition by DCs may be of most relevance especially in the context of infection and transplantation. We have previously published that both immature and mature bone marrow-derived DCs are capable of acquiring MHC:peptide complexes suggesting that this phenomenon may take place in both the peripheral and lymphoid tissues (20). DCs trafficking through infected tissue may well use this pathway to acquire, and subsequently present, MHC:peptide complexes within the lymph nodes. It has been described that lymph node resident CD8{alpha}+ DCs can capture cellular Ags from trafficking CD8{alpha}– DC subsets, such as dermal/interstitial DCs or Langerhans’ cells, and cross-present them to naive CD8+ T cells (16, 38, 43). We have observed, in vitro, that splenic CD8{alpha}+ DCs have the ability to acquire functional MHC:peptide complexes from other DCs pulsed with high concentrations of Ag and stimulate CD8+ T cell proliferation. The efficiency of MHC transfer in stimulating T cells is dictated by the Ag dose, as the level of MHC-peptide acquisition by CD8{alpha}+ cells when the Ag is low is negligible. This result suggests that the level of MHC:peptide acquisition by CD8{alpha}+ cells when Ag concentrations are limiting may not be enough to initiate a CD8+ T cell response. However, if Ag was in excess it is reasonable to suggest, from our data, that this pathway may be involved in T cell activation. Whether such levels can be reached in vivo remains unclear.

Although, CD8{alpha}+ DCs appear to be the main DC subtype involved in priming of CD8+ T cells, non-CD8 DC populations can also be involved (44). We have observed that CD8{alpha}– DCs can acquire MHC:peptide molecules from other DCs and stimulate a CD8+ T cell response at high Ag concentrations.

It is also possible that noninfected DCs acquire MHC:peptide complexes from infected nonhematopoietic cells at the site of infection or in the draining lymph node. This may indeed be the case as we have observed that DCs can acquire MHC:peptide complexes from epithelial cells. Importantly, transferred MHC molecules are fully functional. In agreement with previously published observations, transferred MHC class I:OVA complexes were capable of inducing Ag-specific CD8+ T cell proliferation and IL-2 production in our study even when the Ag density was limited (20). In addition we also have shown that DCs (BMDCs) can acquire functional MHC:peptide molecules from donating DCs infected with virus. Whether this is also the case when the recipient cell is a splenic DC is yet to be tested.

The relative contribution of cross-presentation and cross-dressing in priming CD8+ T cells was also investigated in this study. We observed that Ag presentation by BMDCs and CD8{alpha}– DCs following acquisition of MHC class I:peptide complexes was more efficient at stimulating Ag-specific T cell proliferation when compared with Ag acquired and presented via cross-presentation. The reverse was true for CD8{alpha}+ splenic DCs. The discrepancy between the relative contribution of cross-presentation and cross-dressing in priming CD8+ T cells may reflect a difference in phagocytic capacity of BMDCs as compared with CD8{alpha}+ DCs, or the ability of each DC type to interact with other cells. "Nibbling" is one way in which fragments of plasma membrane are acquired by DCs (45, 46). Plasma membrane can also be acquired in the form of exosome uptake (47). Although BMDCs have been shown to nibble plasma membrane from other cells it may be the case that splenic DCs do not nibble as efficiently. Recently, Segura et al. (48) have shown that CD8{alpha}+ DCs can acquire exosomes bearing functional MHC class II complexes both in vitro and in vivo and stimulate CD4+ T cells.

Although transfer of MHC molecules has been shown in vitro, whether this phenomenon occurs in vivo at levels sufficient to be of biological relevance is still a subject of much debate and continuing investigation. Early experiments suggest that MHC transfer may not occur in vivo. When TCR-transgenic OT-1 T cells, specific for an OVA peptide presented by H-2Kb, were injected into mice transgenic for OVA expressed in the pancreas, the transferred T cells divided vigorously in the draining lymph node. This was presumed to result from the capture and processing of OVA by trafficking DCs. However, if the OVA-transgenic mice were made chimeric with H-2bm1 bone marrow (Kbm1 cannot present the OVA peptide to OT-1 T cells) no OT-1 T cell division was seen. This suggests that the trafficking Kbm1 DCs, did not acquire intact complexes of Kb with OVA peptides from the pancreatic β cells in sufficient quantities to induce OT-1 T cell proliferation (49). However more recent data suggests that MHC transfer does indeed occur in vivo. We have observed MHC transfer in vivo within recombinant IFN-{gamma}-treated mice. Injecting either immature or mature DCs into a recipient animal previously challenged with recombinant IFN-{gamma}, resulted in acquisition of donor MHC molecules by DCs. Trafficking H-2E–/– (B10A.4R) DCs injected into H-2E+ (B10A.2R) recipient mice acquired intact MHC molecules from allogeneic cells in vivo under inflammatory conditions. These acquired complexes are fully functional, in as much as H-2E–/– DCs were able to present the H-Y peptide to H-2E-restricted T cells when purified, by cell sorting, from lymphoid tissue of the recipient mice (20). Whether transfer of MHC class II in this model is via direct cell-to-cell interaction or through production of exosomes is unknown at this point. Indeed how MHC is transferred in vivo has yet to be elucidated. This observation suggests that MHC transfer in vivo may be more efficient under inflammatory conditions as compared with the steady state. This may explain the bone marrow chimera observed, although our in vivo observations involved MHC class II; the transfer of MHC class I under local inflammatory conditions has yet to be addressed.

In conclusion, at this time we do not know whether class I MHC: peptide complex acquisition in vivo plays a role in initiating and or expanding immune responses, nor whether it is as effective at priming T cells in vivo as cross-presentation. Although highly speculative the respective roles of these two mechanisms of Ag presentation may be a question of timing. As one of the major sources of Ag for cross-presentation is uptake of dying cells, it is feasible that MHC:Ag complex acquisition could occur before cell death and cross-presentation. MHC transfer does not involve reprocessing of the acquired Ag and may contribute to the early stages of T cell activation while cross-presentation may predominate thereafter. Transfer of MHC complexes between DCs may be another mechanism to induce T cell responses when phagocytic capacity of DCs has been down-regulated following TLR activation (40). Therefore it is possible that optimal priming of CD8+ T cells results from a combination of cross-dressing and cross-presentation by different DC subtypes. In addition, for DCs with limited cross-presentation capacity, for example the CD8{alpha}– subset (39) acquisition of MHC:peptide complexes maybe a route by which these cells acquire Ag for T cell priming. We are in the process of addressing whether MHC class I transfer occurs in vivo in both transplant and viral models.


   Acknowledgments
 
We thank Dr. Sandra Diebold for helpful discussions.


   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 a grant from the British Heart Foundation. Back

2 Address correspondence and reprint requests to Dr. Robert Lechler, Immunoregulation Laboratories, 5th Floor Thomas Guys House, King’s College London, Guy’s Campus, London SE1 9RT, U.K. E-mail address: robert.lechler{at}kcl.ac.uk Back

3 Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; rAd, recombinant adenovirus. Back

Received for publication September 7, 2007. Accepted for publication June 26, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Belz, G. T., C. M. Smith, D. Eichner, K. Shortman, G. Karupiah, F. R. Carbone, W. R. Heath. 2004. Cutting edge: conventional CD8{alpha}+ dendritic cells are generally involved in priming CTL immunity to viruses. J. Immunol. 172: 1996-2000. [Abstract/Free Full Text]
  2. Norbury, C. C.. 2003. Cross priming or direct priming: is that really the question?. Curr. Opin. Immunol. 15: 82-88. [Medline]
  3. Norbury, C. C., D. Malide, J. J. Gibbs, J. R. Bennink, J. W. Yewdell. 2002. Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo. Nat. Immunol. 3: 265-271. [Medline]
  4. Bevan, M. J.. 1976. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J. Exp. Med. 143: 1283-1286. [Abstract/Free Full Text]
  5. Zinkernagel, R. M.. 2002. On cross-priming of MHC class I-specific CTL: rule or exception?. Eur. J. Immunol. 32: 2385-2392. [Medline]
  6. Mount, A. M., C. M. Smith, F. Kupresanin, K. Stroermer, W. H. Heath, G. T. Beltz. 2008. Multiple dendritic cell populations activate CD4+ T cells after viral stimulation. PLoS ONE 3: e1691
  7. Masson, F., A. M. Mount, N. S. Wilson, G. T. Belz. 2008. Dendritic cells: driving the differentiation programme of T cells in viral infections. Immunol. Cell Biol. 86: 333-342. [Medline]
  8. Heath, W. R., F. R. Carbone. 2001. Cross-presentation in viral immunity and self tolerance. Nat. Rev. Immunol. 1: 126-134. [Medline]
  9. Hertel, L., V. G. Lacaille, H. Strobl, E. D. Mellins, E. S. Mocarski. 2003. Susceptibility of immature and mature langerhans cell-type dendritic cells to infection an immunomodulation by human cytomegalovirus. J. Virol. 77: 7563-7574. [Abstract/Free Full Text]
  10. Moutaftsi, M., A. M. Mehl, L. K. Borysiewicz, Z. Tabi. 2002. Human cytomegalovirus inhibits maturation and impairs function of monocyte-derived dendritic cells. Blood 99: 2913-2921. [Abstract/Free Full Text]
  11. Sigal, L. J., K. L. Rock. 2000. Bone marrow-derived antigen-presenting cells are required for the generation of cytotoxic T lymphocyte responses to viruses and use transporter associated with antigen presentation (TAP)-dependent and -independent pathways of antigen presentation. J. Exp. Med. 192: 1143-1150. [Abstract/Free Full Text]
  12. Sigal, L. J., S. Crotty, R. Andino, K. L. Rock. 1999. Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature 398: 77-80. [Medline]
  13. den Haan, J. M. M., S. M. Lehar, M. J. Bevan. 2000. CD8+ but not CD8– dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192: 1685-1695. [Abstract/Free Full Text]
  14. Schnorrer, P., G. M. N. Behrens, N. S. Wilson, J. L. Pooley, C. M. Smith, D. El-Sukkari, G. Davey, F. Kupresanin, M. Li, E. Maraskovsky, et al 2006. The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture. Proc. Natl. Acad. Sci. USA 103: 10729-10734. [Abstract/Free Full Text]
  15. Pooley, J. L., W. R. Heath, K. Shortman. 2001. Cutting edge: Intravenous soluble antigen is presented to CD4 T cells by CD8– dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells. J. Immunol. 166: 5327-5330. [Abstract/Free Full Text]
  16. Allen, R. S., J. Waithman, S. Bedoui, C. M. Jones, J. A. Vilandangos, Y. Zhan, A. M. Lew, K. Shortman, W. A. Heath, F. A. Carbone. 2006. Migratory dendritic cells transfer antigen to a lymph node resident dendritic cell population for efficient CTL priming. Immunity 25: 153-162. [Medline]
  17. Lin, M. L., Y. Zhan, A. I. Proietto, S. Prato, L. Wu, W. R. Heath, J. A. Villadangos, A. M. Lew. 2008. Selective suicide of cross-presenting CD8+ dendritic cells by cytochrome c injection shows functional heterogeneity within this subset. Proc. Natl. Acad. Sci. USA 105: 3029-3034. [Abstract/Free Full Text]
  18. Datta, S. P., V. Redecke, K. R. Prilliman, K. Takabayashi, M. Corr, T. Tallant, J. DiDonato, R. Dziarski, S. Akira, S. P. Schoenberger, E. Raz. 2003. A sunset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells. J. Immunol. 170: 4102-4110. [Abstract/Free Full Text]
  19. Huang, J., T. Tatsumi, E. Pizzoferrato, N. Vujanovic, W. J. Storkus. 2005. Nitric oxide sensitzes tumor cells to dendritic cell-mediated apoptosis, uptake, and cross-presentation. Cancer Res. 65: 8461-8470. [Abstract/Free Full Text]
  20. Herrera, O. B., D. Golshayan, R. Tibbott, F. Salcido Ochoa, M. J. James, F. M. Marelli-Berg, R. I. Lechler. 2004. A novel pathway of alloantigen presentation by dendritic cells. J. Immunol. 173: 4828-4837. [Abstract/Free Full Text]
  21. Dolan, B. P., K. D. Gibbs, Jr, S. Ostrand-Rosenberg. 2006. Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines. J. Immunol. 176: 1447-1455. [Abstract/Free Full Text]
  22. Dolan, B., K. Gibbs, S. Ostrand-Rosenberg. 2006. Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells. J. Immunol. 177: 6018-6024. [Abstract/Free Full Text]
  23. Game, D. S., N. J. Rogers, R. I. Lechler. 2005. Acquisition of HLA-DR and costimulatory molecules by T cells from allogeneic antigen presenting cells. Am. J. Transplant. 5: 1614-1625. [Medline]
  24. Tanaka, Y., O. Williams, R. Tarazona, A. Wack, T. Norton, D. Kioussis. 1997. In vitro positive selection of {alpha}β TCR transgenic thymocytes by a conditionally immortalised cortical epithelial clone. Int. Immunol. 9: 381-393. [Abstract/Free Full Text]
  25. Gil-Torregrosa, B. C., A. M. Lennon-Dumenil, B. Kessler, P. Guermonprez, H. L. Ploegh, D. Fruci, P. Van Endert, S. Amigorena. 2004. Control of cross-presentation during dendritic cell maturation. Eur. J. Immunol. 34: 398-407. [Medline]
  26. Herrera, O. B., S. Brett, R. I. Lechler. 2002. Infection of mouse bone marrow-derived dendritic cells with recombinant adenovirus vector leads to presentation of encoded antigen by both MHC class I and class II molecules-potential benefits in vaccine design. Vaccine 21: 231-242. [Medline]
  27. Cheng, C., J. C. D. Gall, W.-P. Kong, R. L. Sheet, P. L. Gomez, C. R. King, G. J. Nabel. 2007. Mechanism of Ad5 vaccine immunity and toxicity: fiber shaft targeting of dendritic cells. PLOS Patholog. 3: e25
  28. Porgador, A., J. W. Yewdell, Y. Deng, J. R. Bennink, R. N. Germain. 1997. Localisation, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6: 715-726. [Medline]
  29. Schulz, O., C. Reis e Sousa. 2002. Cross-presentation of cell-associated antigens by CD8a+ dendritic cells is attributable to their ability to internalize dead cells. Immunology 107: 183-189. [Medline]
  30. Smyth, L. A., B. Afzali, J. Tsang, G. Lombardi, R. I. Lechler. 2007. Intercellular transfer of MHC and immunological molecules: molecular mechanisms and biological significance. Am. J. Transplant. 7: 1-8. [Medline]
  31. Schulz, O., S. S. Diebold, M. Chen, T. I. Naslund, M. A. Nolte, L. Alexopouou, Y.-T. Azuma, R. A. Flavell, P. Lijestrom, C. Reis e Sousa. 2005. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature 433: 887-892. [Medline]
  32. Reis e Sousa, C., R. N. Germain. 1995. Major histocompatibility complex class I presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagocytosis. J. Exp. Med. 182: 841-851. [Abstract/Free Full Text]
  33. Stoitzner, P., C. H. Tripp, A. Eberhart, K. M. Price, J. Y. Jung, L. Bursch, F. Ronchese, N. Romani. 2006. Langerhan cells cross-present antigen derived from skin. Proc. Natl. Acad. Sci. USA 103: 7783-7788. [Abstract/Free Full Text]
  34. Klotman, M. E., T. L. Chang. 2006. Defensins in innate antiviral immunity. Nat. Rev. Immunol. 6: 447-456. [Medline]
  35. Proud, D., S. P. Sanders, S. Wiehler. 2004. Human rhinovirus infection induces airway epithelial cells production of human b- defensins. J. Immunol. 172: 4637-4645. [Abstract/Free Full Text]
  36. Biragyn, A., P. A. Ruffini, C. A. Leifer, E. Klyushnenkova, A. Shakhov, O. Chertov, A. K. Shirakawa, J. M. Farber, D. M. Segal, J. J. Oppenheim, L. W. Kwak. 2002. Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 298: 1025-1029. [Abstract/Free Full Text]
  37. Le Borgne, M., N. Etchart, A. Goubier, S. A. Lira, J. C. Sirard, N. van Rooijen, C. Caux, S. Ait-Yahia, A. Vicari, D. Kaiserlian, B. Dubois. 2006. Dendritic cells rapidly recruited into epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell cross-priming in vivo. Immunity 24: 191-201. [Medline]
  38. Belz, G. T., C. M. Smith, L. Kleinert, P. Reading, A. Brooks, K. Shortman, F. R. Carbone, W. R. Heath. 2004. Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc. Natl. Acad. Sci. USA 101: 8670-8675. [Abstract/Free Full Text]
  39. Chung, Y., J.-H. Chang, B.-S. Kin, J.-M. Lee, H.-Y. Kim, C.-Y. Kang. 2007. Anatomic location defines antigen presentation by dendritic cells to T cell in response to intravenous soluble antigen. Eur. J. Immunol. 37: 1453-1462. [Medline]
  40. Wilson, N. S., G. M. Behrens, R. J. Lundie, C. M. Smith, J. Waithman, L. Young, S. P. Forehan, A. Mount, R. J. Steptoe, K. D. Shortman, et al 2006. Systemic activation of dendritic cells by Toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity. Nat. Immunol. 7: 166-172.
  41. Yewdell, J. W., S. M. Haeryfar. 2005. Understanding presentation of viral antigens to CD8+ T cells in vitro: the key to rational vaccine design. Annu. Rev. Immunol. 23: 651-682. [Medline]
  42. Smyth, L. A., O. B. Herrera, D. Golshayan, G. Lombardi, R. I. Lechler. 2006. A novel pathway of antigen presentation by dendritic and endothelial cells: Implications for allorecognition and infectious diseases. Transplantation 82: S15-S18. [Medline]
  43. Carbone, F. R., G. T. Belz, W. R. Heath. 2004. Transfer of antigen between migrating and lymph node resident DCs in peripheral T-cell tolerance and immunity. Trends Immunol. 25: 654-658.
  44. Mount, A. M., C. M. Smith, K. Kupresanin, K. Stoermer, W. R. Heath, G. T. Belz. 2008. Muktiple dendritic cell populations activate CD4+ T cells after viral stimulation. PLoS ONE 3: 1-10.
  45. Harshyne, L. A., S. C. Watkins, A. Gambotto, S. M. Barratt-Boyes. 2001. Dendritic cells acquire antigens from live cells for cross-presentation to CTL. J. Immunol. 166: 3717-3723. [Abstract/Free Full Text]
  46. Harshyne, L. A., M. I. Zimmer, S. C. Watkins, S. M. Barratt-Boyes. 2003. A role for class A scavenger receptor in dendritic cell nibbling from live cells. J. Immunol. 170: 2302-2309. [Abstract/Free Full Text]
  47. Morelli, A. E., A. T. Larregina, W. J. Shufesky, M. L. Sullivan, D. B. Stolz, G. D. Papworth, A. F. Zahorchak, A. J. Logar, Z. Wang, S. C. Watkins, et al 2004. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood 104: 3257-3266. [Abstract/Free Full Text]
  48. Segura, E., C. Guerin, N. Hogg, S. Amigorena, C. Thery. 2007. CD8+ dendritic cells use LFA-1 to capture MHC-peptide complexes from exosomes in vivo. J. Immunol. 179: 1489-1496. [Abstract/Free Full Text]
  49. Kurts, C., W. Heath, F. Carbone, J. Allison, J. Miller, H. Kosaks. 1996. Constitutive Class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 184: 923-930. [Abstract/Free Full Text]



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