HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Belz, G. T. Articles by Heath, W. R. Search for Related Content PubMed PubMed Citation Articles by Belz, G. T. Articles by Heath, W. R.

CD36 Is Differentially Expressed by CD8⁺ Splenic Dendritic Cells But Is Not Required for Cross-Presentation In Vivo¹

Gabrielle T. Belz^*, David Vremec^*, Maria Febbraio, Lynn Corcoran^*, Ken Shortman^*, Francis R. Carbone and William R. Heath^2,*

^* Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, and Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; and Department of Medicine, Division of Hematology/Oncology, Weill Medical College of Cornell University, New York, NY 10021

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cross-presentation allows the processing of Ags from donor cells into the MHC class I presentation pathway of dendritic cells (DCs). This is important for the generation of cytotoxic T cell immunity and for induction of self tolerance. Apoptotic cells are reported to be efficient targets for cross-presentation, and in vitro studies using human DCs have implicated CD36 in their capture. In support of a role for CD36 in cross-presentation, we show that this molecule is differentially expressed by CD8⁺ splenic DCs, which previously have been identified as responsible for cross-presentation in the mouse. Three different cross-presentation models were examined for their dependence on CD36. These included cross-priming to OVA-coated spleen cells and cross-tolerance to OVA transgenically expressed in the pancreatic islet cells under constitutive conditions or during cell destruction. In these models, CD36 knockout DCs were equivalent to wild-type DCs in their capacity to cross-present either foreign or self Ags, indicating that CD36 is not essential for cross-presentation of cellular Ags in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cross-presentation is a process whereby dendritic cells (DCs)³ capture exogenous Ags, often of cellular origin, and process them into the MHC class I pathway. For self Ags, cross-presentation can be used to generate class I-restricted epitopes for stimulation of the deletion of autoreactive CD8⁺ T cells (1, 2). This has been referred to as cross-tolerance and may be important in the maintenance of both central (2) and peripheral (1) tolerance. For foreign Ags, cross-presentation can result in the generation of epitopes that are used to stimulate cytotoxic T cell immunity (3). Such cross-priming is able to generate cytotoxic T cell responses that can lyse virus-infected cells or cancerous tissue (4, 5, 6, 7). Although cellular Ags are commonly targeted for cross-presentation, there is limited knowledge of how they enter the cross-presentation pathway (8, 9).

One way Ags have been shown to access the cross-presentation pathway is via the capture of apoptotic cells (10, 11). Albert et al. (11) reported that macrophages induced to undergo apoptosis as a result of influenza infection were efficiently captured by DCs, and their influenza-associated Ags cross-presented on class I MHC molecules of the DCs. In an extension of these in vitro studies, these same authors investigated the molecular requirements for the capture of apoptotic cells (10). These studies implicated the integrin _v5 and CD36 as potentially important molecules in the binding of apoptotic cells for cross-presentation. CD36 is thought to mediate its effect by cooperating with _v5 on the DC surface, binding soluble thrombospondin-1, which forms a molecular bridge to the apoptotic cell (12, 13, 14). However, the importance of either apoptosis or molecules associated with the binding of apoptotic cells in cross-presentation in vivo for either cross-priming or cross-tolerance has not been addressed. In this report, we examine the expression of CD36 on DCs in vivo and determine its role in cross-priming and cross-tolerance.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

All mice were bred and maintained at the Walter and Eliza Hall Institute for Medical Research (Parkville, Victoria, Australia). OT-I, RIP-mOVA. bml, and RIP-OVA^low transgenic mice have been described previously (1, 15, 16). CD36^-/- mice were generated and backcrossed onto the C57BL/6 (B6) background for four generations (17). All experiments were done in compliance with institutional guidelines approved by the Animal Ethics Committee and Institutional Biosafety Committee of The Walter and Eliza Hall Institute of Medical Research and Royal Melbourne Hospital.

OVA-specific cross-priming

OVA-specific cross-priming was achieved by coating 2 x 10⁸ irradiated (1000 cGy) H-2^bm1 splenocytes with 1 ml of OVA protein (10 mg/ml; Sigma-Aldrich, St. Louis, MO) in HEPES-buffered Eagle’s medium (HEM) at 37°C for 10 min. Cells were then washed twice in HEM containing 2.5% FCS (HF2.5), filtered through nylon mesh to remove aggregates, and counted. Recipient mice were injected with 2 x 10⁷ cells per mouse i.v. in 0.5 ml of HEM2.5.

Activation of OT-I cells

OT-I cells were activated by culturing 10⁷ OT-I spleen cells for 5 days in 30 ml of complete medium with 10⁷ irradiated (1500 cGy) II-mOVA spleen cells, which expressed a membrane-bound form of OVA under the control of the class II (I-E) promoter.

CFSE labeling of OT-I CD8⁺ T cells and adoptive transfer

Preparation and adoptive transfer of OT-I cells, CFSE labeling, and analysis on a FACScan (BD Biosciences, Mountain View, CA) were conducted as described previously (1). Cells were stained with anti-CD8-PE from Caltag Laboratories (Burlingame, CA). Dead cells were excluded by propidium iodide (PI) staining.

Generation of bone marrow chimeras

To generate bone marrow chimeras, adult mice were lethally irradiated by two doses of 550 cGy 3 h apart and reconstituted with 5 x 10⁶ T cell-depleted bone marrow cells from B6 or CD36^-/- mice. The next day, mice were injected i.p. with 100 µl of T24 (anti-Thy-1) ascites to deplete radioresistant T cells. These mice were left for at least 8 wk before use.

Isolation of DC populations

Ex vivo isolation of DC populations was undertaken as described by Vremec et al. (18). Spleen fragments were digested for 20 min at room temperature with collagenase-DNase and then treated for 5 min with EDTA to disrupt T cell-DC complexes. Light density cells were enriched by centrifugation on a 1.077 g/cm³ Nycodenz layer (Nycomed, Oslo, Norway) for 10 min at 1700 x g. Non-DCs were depleted by incubating in optimized concentrations of mAbs (anti-CD3 (KT3.1.1), anti-Thy1 (T24/31.7), anti-B220 (RA36B2), anti-GR-1 (RB68C5), and anti-erythrocyte (TER119)) and then removing the Ab-binding cells with anti-rat Ig-coupled magnetic beads (Dynabeads; Dynal Biotech, Oslo, Norway). The remaining cells were stained with anti-CD11c-Alexa⁵⁹⁴ (N418), anti-CD8-Cy5 (YTS-169.4), anti-CD4-PE (GK1.5), and anti-CD36 (clone 63) (Cascade Bioscience, Winchester, MA) and detected by anti-mouse IgA-FITC (Caltag Laboratories). PI was included in the final wash (after immunostaining) at 1 µg/ml to label dead cells. PI-positive and autofluorescent cells were gated out in the FL5 channel, and only cells displaying characteristic DC forward and side scatter were accepted. CD11c⁺ DCs were then divided into subpopulations on the basis of CD4 and CD8 expression, and staining for CD36 was determined.

Capture of dying cells

B6 or CD36^-/- DC lines, derived as previously described (19), were labeled red using PKH26-GL (Sigma-Aldrich). The splenocytes were collected in HF2.5 and teased apart, and debris was allowed to sediment over an FCS cushion for 7 min at room temperature. The liquid above the FCS was recovered, and cells were washed and then resuspended in 10 ml of HF2.5 and placed on ice for irradiation (1500 rad). Cells were then labeled with CFSE and washed twice in HF2.5, and then 10⁷ cells were placed into a 60-mm petri dish in 5 ml of HF2.5 at 37°C for 6 h to allow cell death to occur. Dying spleen cells were then collected, washed twice in warmed medium (37°C, DMEM containing 10% FCS, 2-ME, glutamine, and antibiotics), counted, and resuspended in DC culture medium (DMEM containing 10% FCS, 2% GM-CSF, 30% 3T3 medium, glutamine, and 2-ME) for the assay. DCs and dying cells were cocultured for 3 h in a 24-well plate (Falcon; BD Biosciences, Franklin Lakes, NJ), each at 1–2.5 x 10⁵ cells/ml in 2 ml. These cells were recovered by brief exposure to 5 mM EDTA in balanced salt solution and then analyzed by flow cytometry.

Assessment of deletion of OT-I cells

This was achieved as previously described (1). Briefly, recipient mice were generated by reconstituting lethally irradiated RIP-mOVA or negative littermate mice on a bm1 background, with either B6 or CD36^-/- bone marrow. After 10 wk, chimeric mice were injected with 5 x 10⁶ OT-I cells and then left for an additional 8 wk before deletion was assessed. To examine deletion, cells were pooled from the lymph nodes (LNs; inguinal, brachial, axillary, sacral, cervical, and mesenteric) and spleen of each chimeric mouse. They were then stained using anti-CD8-PE (Caltag Laboratories), anti-V5.1/5.2-FITC (MR9-4), and anti-V2-biotin (B20.1) followed by streptavidin-Tricolor (Caltag Laboratories). Live gates were set on lymphocytes by forward and side scatter profiles. Analysis was done on a FACScan (BD Biosciences). A total of 10,000–20,000 live cells were collected for analysis. OT-I cells were identified as V2⁺V5⁺CD8⁺. The proportion of endogenous V2⁺V5⁺CD8⁺ cells (<2%) was assessed by examining mice that did not receive OT-I cells. This value was subtracted from the values derived from OT-I recipients as described (1).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Splenic DC subpopulations differentially express CD36

It recently has been revealed that CD8⁺ DCs are responsible for in vivo cross-presentation of cell-associated (20) or soluble (21) Ags. Furthermore, apoptotic cells have been reported to be an important source of cell-associated Ags for cross-presentation (11). Because CD36 has been implicated in the capture of apoptotic cells, we examined its expression on splenic DC subsets (Fig. 1). This revealed that CD36 was highly expressed by the CD8⁺ subset of DCs and that only very low levels could be detected on the CD4⁺ and the CD4^-CD8^- subsets. The differential expression of CD36 by CD8⁺ splenic DCs was consistent with the idea that apoptotic cells were the prime source of cell-associated material for cross-presentation.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. CD8+ splenic DCs express CD36. Splenic DCs from either wild-type B6 (bold line) or CD36 knockout (gray line) mice were purified as described in Materials and Methods and then stained for expression of CD36, CD11c, CD4, and CD8. Cells were gated on CD11c+ and further gated on CD4+CD8- cells (uper panel), CD4-CD8- cells (middle panel), or CD4-CD8+ cells (lower panel) and examined for CD36 expression.

Because CD36 was highly expressed by CD8⁺ DCs, this warranted exploration of its role in cross-priming in vivo. To do this, we examined the capacity of transgenic CD8⁺ T cells (OT-I) to proliferate in response to cross-priming with OVA-coated irradiated splenocytes in wild-type or CD36 knockout mice. In this case, OVA is not presented by the donor cells, but must be captured (in a cell-associated form) by host CD8⁺ splenic DCs and cross-presented to responding T cells (20, 22, 23). OT-I cells were labeled with CFSE and adoptively transferred into either wild-type B6 mice or CD36 knockout mice. These two groups were then primed with OVA-coated irradiated spleen cells, and 3 days later their spleens were harvested and single cell suspensions were examined by flow cytometry. OT-I cells proliferated extensively in wild-type and CD36 knockout mice, indicating that expression of CD36 on DCs was not essential for cross-priming (Fig. 2). This was the case even when mice were stimulated with irradiated spleen cells coated with 10-fold less OVA, which represents a suboptimal amount of Ag (data not shown).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Proliferation of CFSE-labeled OT-I cells after cross-priming. CFSE-labeled OT-I cells (2 x 106) were adoptively transferred into B6 (A and B) or CD36-/- (C) recipients. One day later, mice were injected i.v. with 2 x 107 OVA-coated (10 mg/ml) irradiated H-2bm1 splenocytes (B and C) or were left unprimed (A). Three days later, their spleens were harvested and cells were analyzed by flow cytometry. Mice that did not receive Ag did not induce proliferation (A). Histograms are gated on CD8+CFSE+ cells.

Cross-presentation can be associated with either priming to foreign Ags (cross-priming) or tolerance to self (cross-tolerance). Using mice expressing OVA in the pancreas under the control of the RIP (RIP-mOVA mice), we have previously shown that tissue-associated OVA is captured by host DCs and constitutively cross-presented to autoreactive CD8⁺ T cells in the pancreatic draining LN (24, 25). This stimulates the proliferation of these autoreactive CD8⁺ T cells but eventually leads to their deletion (1), thus maintaining self tolerance. To determine whether the expression of CD36 was necessary for the constitutive cross-presentation of self Ags by DCs, cross-presentation was examined in RIP-mOVA mice reconstituted with bone marrow from either wild-type or CD36 knockout mice. The DC compartment of these mice would be reconstituted from the donor bone marrow and therefore would be of either wild-type or CD36 knockout phenotype, depending on its source. Reconstitution with bone marrow cells expressing an MHC haplotype that is unable to present OVA previously has been shown to prevent cross-presentation (1), indicating that this method efficiently replaces host APCs. Analysis of proliferation of OT-I cells in the pancreatic draining LN 3 days after adoptive transfer revealed that cross-presentation of OVA occurred normally in the absence of CD36 (Fig. 3). Thus, constitutive cross-presentation of self Ag does not require CD36 expression by DCs.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Bone marrow-derived APCs lacking CD36 efficiently cross-present self Ag. CFSE-labeled OT-I cells (2 x 106) were transferred to B6 (A) or RIP-mOVA (B and C) chimeric mice. These chimeras were of a bm1 background and had been grafted with either B6 (A and B) or CD36-/- (C) bone marrow. After 3 days, pancreatic LN cells were recovered and single cells were analyzed by flow cytometry. Profiles were gated on CD8+CFSE+ cells.

Although the above data had not revealed a role for CD36 in cross-presentation, neither of the above forms of cross-presentation had been formally shown to require apoptosis. Thus, it seemed possible that cross-presentation might not always target apoptotic cells and only when it does would CD36 expression be essential. To examine this issue, we used a model (RIP-OVA^low mice) in which effective cross-presentation of self Ags requires the destruction (and apoptosis) of self tissues (26). In RIP-OVA^low mice, OVA is expressed in the pancreas at a lower level than in RIP-mOVA animals and is only cross-presented in the draining pancreatic LN when these mice are first adoptively transferred with activated OT-I cells. These activated OT-I cells kill OVA-expressing islet cells, and this provides Ag for cross-presentation in the pancreatic LN. To test the role of CD36 in this model, RIP-OVA^low mice were lethally irradiated and then reconstituted with either wild-type or CD36 knockout bone marrow to generate the respective DCs. After sufficient time for reconstitution of their DC populations, these chimeric mice were injected with activated OT-I cells, which then caused the destruction of islet cells. This destruction leads to cross-presentation of islet Ags, which can be assessed by injection of naive CFSE-labeled OT-I cells. Proliferation of these cells to cross-presented islet-derived Ags is then examined 3 days later. This experiment showed that when cross-presentation of islet Ags was initiated by tissue damage, OT-I cells proliferated equally well to Ags presented by wild-type or CD36 knockout DCs (Fig. 4). As an Ag-specific control, some mice were transferred with CFSE-labeled normal B6 T cells (instead of OT-I cells). As expected, these cells did not proliferate in response to cross-presented islet OVA.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Tissue destruction leads to cross-presentation of OVA even in the absence of CD36. RIP-OVAlow mice were lethally irradiated and reconstituted with either B6 (A and B) or CD36-/- (C and D) bone marrow. Eight weeks later, all mice were injected i.v. with 5 x 106 activated OT-I cells (see Materials and Methods) to cause islet destruction. Four days later, mice were injected with 2 x 106 CFSE-labeled naive OT-I cells (B and D) or normal B6 CD8+ cells (A and C). After an additional 3 days, pancreatic LN cells were harvested and single cells were analyzed by flow cytometry. Histograms were gated on CD8+CFSE+ cells. No proliferation would be detected in the pancreatic LN if activated OT-I cells were not first injected into these recipients (26 ).

Previously we have shown that cross-presentation of self Ags leads to the deletion of autoreactive CTLs (1). Although the above data did not reveal a role for CD36 in the cross-presentation of self Ags, it was possible that CD36 binding to apoptotic cells might stimulate DCs to provide signals essential for the deletion process. Thus, although Ag capture might not require CD36, other signals from the apoptotic cell to the DC might need this molecule. To examine the influence of CD36 on deletion by cross-tolerance, we tested the survival of OT-I cells in RIP-mOVA mice that contained either wild-type or CD36 knockout DCs. RIP-mOVA mice on a bm1 background were irradiated and reconstituted with either B6 bone marrow (B6RIP-mOVA) or CD36^-/- bone marrow (CD36^-/-RIP-mOVA). RIP-mOVA mice on a bm1 background were used to ensure that islet cells could not be destroyed by OT-I cells (K^bm1 cannot present OVA to OT-I cells). Ten weeks after reconstitution, 5 x 10⁶ OT-I cells were transferred into chimeric recipients and examined for survival after an additional 8 wk (Fig. 5). In this case, deletion proceeded normally in the absence of CD36.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Deletion of cross-presented self Ags is unaffected by the absence of the CD36 molecule on bone marrow-derived cells. Bone marrow cells from B6 or CD36-deficient mice were grafted into RIP- mOVA.m1 mice and nontransgenic littermates. Ten weeks later, 5 x 106 OT-I cells were adoptively transferred, and after 8 wk the number of V2+V5+CD8+ T cells in the LN and spleen was determined by flow cytometry. Individuals () and means (horizontal bars) for each group are shown.

So far, we had been unable to detect an essential role for CD36 in various models of cross-presentation. One possibility was that despite testing an array of models, none have relied on apoptosis of target tissue for cross-presentation. This is unlikely but possible because it has not been formally proven that cross-presentation in vivo ever requires apoptotic cell targets, even when it does rely on tissue damage as in Fig. 4. With this in mind, we decided to ask whether CD36 was essential for the uptake of dying cells by murine DCs. To address this, we used long-term immature DC lines generated by the in vitro culture of spleen cells with NIH/3T3 supernatant containing GM-CSF as described (19). DCs derived from B6 mice expressed CD36, whereas those from CD36^-/- mice did not (data not shown). When these cells were cultured for 3 h with dying spleen cells, similar uptake was seen for wild-type and knockout DCs (Fig. 6). This experiment showed 22% uptake by wild-type cells and 18% by CD36^-/- cells. In two other experiments, these values were 34 vs 33% and 28 vs 26%, respectively. Thus, CD36 did not appear to play an essential role in the uptake of dying cells by these DCs.

View larger version (40K): [in this window] [in a new window]   FIGURE 6. CD36 expression is not essential for capture of dying spleen cells. To examine capture of dying cells, splenic-derived DC lines (19 ) from B6 (B and C) or CD36-/- (D) mice were labeled with PKH26-GL and cultured at a ratio of 1:1 with CFSE-labeled dying splenocytes (A). Uptake by B6 (C) or CD36-/- (D) DC lines was analyzed after a 3-h culture with dying spleen cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using a classical model for cross-priming (27), i.e., i.v. administered OVA-coated spleen cells, we were unable to show a role for CD36 in the capture and presentation of exogenous cell-associated Ag. In one preliminary experiment, we also failed to see any effect of CD36 on the strength of the CTL response by a normal repertoire of T cells when induced by such cross-priming (data not shown). Thus, neither Ag cross-presentation nor signaling involved in the priming process is affected by CD36. Similarly, we found no role for CD36 in the capture and cross-presentation of tissue-associated OVA in transgenic mice where OVA was expressed as a self Ag in the pancreas and kidney. This was the case even if cross-presentation was mediated by first causing tissue destruction, which we previously have shown is associated with the generation of apoptotic cells. Thus, in our three quite different models of cross-presentation, one leading to cross-priming and the other two involved in cross-tolerance, we were unable to identify a role for CD36.

One alternative possibility was that CD36 was required for delivery of an anti-inflammatory signal to the DC, enabling it to induce the deletion of self-reactive CTLs during cross-tolerance. This possibility arose from the reported capacity of CD36 signaling to deliver anti-inflammatory signals to macrophages that have phagocytosed apoptotic material (28). Again, however, CD36 expression was not essential to induce the deletion of self-reactive CTLs by cross-tolerance. These data strongly suggest that CD36 is not essential for cross-presentation in vivo.

The high expression of CD36 by CD8⁺ splenic DCs but not other DC subsets, however, does suggest that CD36 has a role in the function of this subset. Because these cells are the major DC subset thought to be responsible for cross-presentation (20), it seems logical to anticipate some role for CD36 in this process. Therefore, we are left with three main alternatives: 1) CD36 has a redundant function that can be compensated in knockout mice, 2) it plays a role in cross-presentation but not in the systems we tested, or 3) it has an as yet unidentified function in the CD8⁺ subset of DCs unrelated to cross-presentation. With regard to the first possibility, there are several pathways by which apoptotic cells may be captured, and in the studies using human DCs (10), inhibition of CD36 binding only partially impaired apoptotic cell uptake. To uncover function in the case of redundancy, we attempted to increase the sensitivity of our model by reducing the Ag concentration to a suboptimal level when cross-priming with OVA-coated spleen cells. In this case, however, we still failed to identify a role for CD36 (data not shown). Therefore, it is difficult to support the conclusion that redundancy hindered our ability to observe function. In terms of the limitations of the number of cross-presentation models we have tested, we cannot exclude the possibility that there are as yet other types of cross-presentation that do depend on CD36. However, our studies have been quite extensive, examining both cross-priming and cross-tolerance under conditions in which apoptotic cells clearly represent a component of available Ag (irradiated spleen cells in the case of OVA-coated spleen and killed cells for one of the cross-tolerance models). Thus, we are left with the view that CD36 provides an undefined function in the CD8⁺ splenic subset of DCs that is not related directly to cross-presentation.

With respect to the differential expression of CD36 on CD8⁺ DCs and its potential relationship to cross-presentation, it is worth pointing out that such expression may be developmentally related (rather than subset specific). In fact, analysis of DC subsets from Flt-3 ligand-treated mice indicated reasonable expression of CD36 on double-negative DCs (data not shown). Furthermore, treatment of normal B6 mice with LPS, known to stimulate the cross-presentation of soluble OVA by double-negative DCs, did not induce CD36 expression on this subset (data not shown).

It should be stressed that, although CD36 has been suggested to be involved in human DC cross-presentation, the only evidence that it is associated with this process is derived from an indirect experiment examining the uptake of apoptotic cells. Thus, it is only implicated to be involved in cross-presentation because in this system apoptosis was shown to be important for cross-presentation (10). Because of this indirect link, our failure to observe a role for CD36 in cross-presentation could be concluded to be because cross-presentation in our models does not involve uptake of apoptotic cells. Thus, instead of examining the role of CD36 in cross-presentation by DCs, we decided to directly address whether CD36 expression affected DC uptake of dying cells (Fig. 6). To our surprise, we were unable to observe a role for CD36 in this process.

Our studies demonstrate that although the subset of DCs reported to be responsible for cross-priming, i.e., the CD8⁺ DCs, expresses high levels of CD36, this molecule is not required for either cross-priming or cross-tolerance in vivo. Furthermore, in vitro analysis showed that capture of apoptotic material occurred in the absence of CD36 expression by DCs, indicating that, at least in the mouse, it is also not essential for this process.

	Acknowledgments

 
We thank Jane Langley and Tatiana Banjanin for technical assistance.

	Footnotes

 
¹ This work was supported by the National Health and Medical Research Council of Australia and the National Institutes of Health. W.R.H. is supported by a Howard Hughes Medical Institute International Research Fellowship. G.T.B. is a C. J. Martin Fellow of the National Health and Medical Research Council of Australia.

² Address correspondence and reprint requests to Dr. William R. Heath, Immunology Division, Walter and Eliza Hall Institute, Post Office Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. E-mail address: heath{at}wehi.edu.au

³ Abbreviations used in this paper: DC, dendritic cell; HEM, HEPES-buffered Eagle’s medium; PI, propidium iodide; LN, lymph node.

Received for publication January 22, 2002. Accepted for publication April 5, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kurts, C., H. Kosaka, F. R. Carbone, J. F. Miller, W. R. Heath. 1997. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8⁺ T cells. J. Exp. Med. 186:239.[Abstract/Free Full Text]
2. Merkenschlager, M., M. O. Power, H. Pircher, A. G. Fisher. 1999. Intrathymic deletion of MHC class I-restricted cytotoxic T cell precursors by constitutive cross-presentation of exogenous antigen. Eur. J. Immunol. 29:1477.[Medline]
3. 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.[Abstract/Free Full Text]
4. 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.[Medline]
5. Arrode, G., C. Boccaccio, J. Lule, S. Allart, N. Moinard, J. P. Abastado, A. Alam, C. Davrinche. 2000. Incoming human cytomegalovirus pp65 (UL83) contained in apoptotic infected fibroblasts is cross-presented to CD8⁺ T cells by dendritic cells. J. Virol. 74:10018.[Abstract/Free Full Text]
6. Chiodoni, C., P. Paglia, A. Stoppacciaro, M. Rodolfo, M. Parenza, M. P. Colombo. 1999. Dendritic cells infiltrating tumors cotransduced with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand genes take up and present endogenous tumor-associated antigens, and prime naive mice for a cytotoxic T lymphocyte response. J. Exp. Med. 190:125.[Abstract/Free Full Text]
7. Ochsenbein, A. F., S. Sierro, B. Odermatt, M. Pericin, U. Karrer, J. Hermans, S. Hemmi, H. Hengartner, R. M. Zinkernagel. 2001. Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 411:1058.[Medline]
8. Yewdell, J. W., C. C. Norbury, J. R. Bennink. 1999. Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8⁺ T cell responses to infectious agents, tumors, transplants, and vaccines. Adv. Immunol. 73:1.[Medline]
9. Heath, W. R., F. R. Carbone. 2001. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19:47.[Medline]
10. Albert, M. L., S. F. Pearce, L. M. Francisco, B. Sauter, P. Roy, R. L. Silverstein, N. Bhardwaj. 1998. Immature dendritic cells phagocytose apoptotic cells via _v5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.[Abstract/Free Full Text]
11. Albert, M. L., B. Sauter, N. Bhardwaj. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86.[Medline]
12. Savill, J., N. Hogg, Y. Ren, C. Haslett. 1992. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J. Clin. Invest. 90:1513.
13. Rubartelli, A., A. Poggi, M. R. Zocchi. 1997. The selective engulfment of apoptotic bodies by dendritic cells is mediated by the _v3 integrin and requires intracellular and extracellular calcium. Eur. J. Immunol. 27:1893.[Medline]
14. Fadok, V. A., J. S Savill, C. Haslett, D. L. Bratton, D. E. Doherty, P. A. Campbell, P. M. Henson. 1992. Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J. Immunol. 149:4029.[Abstract]
15. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76:17.[Medline]
16. Kurts, C., R. M. Sutherland, G. Davey, M. Li, A. M. Lew, E. Blanas, F. R. Carbone, J. F. Miller, W. R. Heath. 1999. CD8⁺ T cell ignorance or tolerance to islet antigens depends on antigen dose. Proc. Natl. Acad. Sci. USA 96:12703.[Abstract/Free Full Text]
17. Febbraio, M., N. A. Abumrad, D. P. Hajjar, K. Sharma, W. Cheng, S. F. Pearce, R. L. Silverstein. 1999. A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism. J. Biol. Chem. 274:19055.[Abstract/Free Full Text]
18. Vremec, D., J. Pooley, H. Hochrein, L. Wu, K. Shortman. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164:2978.[Abstract/Free Full Text]
19. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V. S. Zimmermann, J. Davoust, P. Ricciardi-Castagnoli. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 185:317.[Abstract/Free Full Text]
20. den Haan, J. 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.[Abstract/Free Full Text]
21. 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.[Abstract/Free Full Text]
22. Bennett, S. R., F. R. Carbone, F. Karamalis, J. F. Miller, W. R. Heath. 1997. Induction of a CD8⁺ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⁺ T cell help. J. Exp. Med. 186:65.[Abstract/Free Full Text]
23. Carbone, F. R., M. J. Bevan. 1990. Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo. J. Exp. Med. 171:377.[Abstract/Free Full Text]
24. Kurts, C., M. Cannarile, I. Klebba, T. Brocker. 2001. Dendritic cells are sufficient to cross-present self-antigens to CD8 T cells in vivo. J. Immunol. 166:1439.[Abstract/Free Full Text]
25. Kurts, C., W. R. Heath, F. R. Carbone, J. Allison, J. F. Miller, H. Kosaka. 1996. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 184:923.[Abstract/Free Full Text]
26. Kurts, C., J. F. Miller, R. M. Subramaniam, F. R. Carbone, W. R. Heath. 1998. Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction. J. Exp. Med. 188:409.[Abstract/Free Full Text]
27. Carbone, F. R., N. A. Hosken, M. W. Moore, M. J. Bevan. 1989. Class I MHC-restricted cytotoxic responses to soluble protein antigen. Cold Spring Harbor Symp. Quant. Biol. 1:551.
28. Voll, R. E., M. Herrmann, E. A. Roth, C. Stach, J. R. Kalden, I. Girkontaite. 1997. Immunosuppressive effects of apoptotic cells. Nature 390:350.[Medline]

This article has been cited by other articles:

				M. Nakayama, H. Akiba, K. Takeda, Y. Kojima, M. Hashiguchi, M. Azuma, H. Yagita, and K. Okumura Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation Blood, April 16, 2009; 113(16): 3821 - 3830. [Abstract] [Full Text] [PDF]

				S. Bedoui, S. Prato, J. Mintern, T. Gebhardt, Y. Zhan, A. M. Lew, W. R. Heath, J. A. Villadangos, and E. Segura Characterization of an Immediate Splenic Precursor of CD8+ Dendritic Cells Capable of Inducing Antiviral T Cell Responses J. Immunol., April 1, 2009; 182(7): 4200 - 4207. [Abstract] [Full Text] [PDF]

				H. Hemmi, J. Idoyaga, K. Suda, N. Suda, K. Kennedy, M. Noda, A. Aderem, and R. M. Steinman A New Triggering Receptor Expressed on Myeloid Cells (Trem) Family Member, Trem-Like 4, Binds to Dead Cells and Is a DNAX Activation Protein 12-Linked Marker for Subsets of Mouse Macrophages and Dendritic Cells J. Immunol., February 1, 2009; 182(3): 1278 - 1286. [Abstract] [Full Text] [PDF]

				E. Tagliani, P. Guermonprez, J. Sepulveda, M. Lopez-Bravo, C. Ardavin, S. Amigorena, F. Benvenuti, and O. R. Burrone Selection of an Antibody Library Identifies a Pathway to Induce Immunity by Targeting CD36 on Steady-State CD8{alpha}+ Dendritic Cells J. Immunol., March 1, 2008; 180(5): 3201 - 3209. [Abstract] [Full Text] [PDF]

				C. Maas, S. Hermeling, B. Bouma, W. Jiskoot, and M. F. B. G. Gebbink A Role for Protein Misfolding in Immunogenicity of Biopharmaceuticals J. Biol. Chem., January 26, 2007; 282(4): 2229 - 2236. [Abstract] [Full Text] [PDF]

				A. Puig-Kroger, A. Dominguez-Soto, L. Martinez-Munoz, D. Serrano-Gomez, M. Lopez-Bravo, E. Sierra-Filardi, E. Fernandez-Ruiz, N. Ruiz-Velasco, C. Ardavin, Y. Groner, et al. RUNX3 Negatively Regulates CD36 Expression in Myeloid Cell Lines J. Immunol., August 15, 2006; 177(4): 2107 - 2114. [Abstract] [Full Text] [PDF]

				M. Skoberne, S. Somersan, W. Almodovar, T. Truong, K. Petrova, P. M. Henson, and N. Bhardwaj The apoptotic-cell receptor CR3, but not {alpha}vbeta5, is a regulator of human dendritic-cell immunostimulatory function Blood, August 1, 2006; 108(3): 947 - 955. [Abstract] [Full Text] [PDF]

				S. Burgdorf, V. Lukacs-Kornek, and C. Kurts The Mannose Receptor Mediates Uptake of Soluble but Not of Cell-Associated Antigen for Cross-Presentation. J. Immunol., June 1, 2006; 176(11): 6770 - 6776. [Abstract] [Full Text] [PDF]

				K. M. Kerksiek, F. Niedergang, P. Chavrier, D. H. Busch, and T. Brocker Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo Blood, January 15, 2005; 105(2): 742 - 749. [Abstract] [Full Text] [PDF]

				A. D. Edwards, D. Chaussabel, S. Tomlinson, O. Schulz, A. Sher, and C. Reis e Sousa Relationships Among Murine CD11chigh Dendritic Cell Subsets as Revealed by Baseline Gene Expression Patterns J. Immunol., July 1, 2003; 171(1): 47 - 60. [Abstract] [Full Text] [PDF]

				O. Schulz, D. J. Pennington, K. Hodivala-Dilke, M. Febbraio, and C. Reis e Sousa CD36 or {alpha}v{beta}3 and {alpha}v{beta}5 Integrins Are Not Essential for MHC Class I Cross-Presentation of Cell-Associated Antigen by CD8{alpha}+ Murine Dendritic Cells J. Immunol., June 15, 2002; 168(12): 6057 - 6065. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Belz, G. T. Articles by Heath, W. R. Search for Related Content PubMed PubMed Citation Articles by Belz, G. T. Articles by Heath, W. R.