This work is supported by the Takasaki University of Health and Welfare.

1. Growing Cells and Addition of Exogenous Antigens
<ol>
	<li>Prepare bOVA using a biotin-protein labeling kit following the manufacturer&#39;s protocol. 
	NOTE: Ordinarily, bOVA contains 2 M biotin per 1 M OVA on average.</li>
	<li>Grow DC2.4 cells in RPMI-1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin-streptomycin, 55 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.5), and 10% fetal calf serum (hereafter RPMI) at 37 &#176;C in 5% CO2 in a hu...

<ol>
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In previous studies of CP, the incorporated exogenous antigens accumulated in the restricted area of the late endosome or ER by immunofluorescent microscopy16,30,31,32. It is estimated that ERAD-like transport and processing of exogenous antigens are carried out in these specialized areas of the ER or late endosome, as the cellular compartment was identified by sucrose or iodixanol density grad...

The MHC I molecules are expressed on the surface of all nucleated cells, with short antigenic peptides derived from endogenous antigens, which are processed by the ubiquitin-proteasome system in the cytosol1. After processing, antigenic peptides are transported into the endoplasmic reticulum (ER) lumen by the peptide transporter TAP. In the ER lumen, a series of specific chaperones assist the peptide loading and the correct folding of the MHC I complex. This series of molecules is called the peptide-loading complex (PLC), indicating that the ER is a central compartment for peptide loading upon MHC I2. After peptide loading, the MHC I molecules are transported to the cell surface and play a key role in the adaptive immune system as self-markers, and enables the CD8+ cytotoxic T lymphocytes (CTLs) to detect cancer cells or infectious agents by antigenic peptides from non-self proteins3.
In antigen presenting cells (APC), antigenic peptides from exogenous antigens are also presented upon MHC I4,5,6,7,8 via CP, which is mainly carried out by DCs9,10,11. CP is essential both for the activation of na&#239;ve CD8+ T cells and memory CD8+ T cells into anti-infectious and anti-tumoral CTLs12,13, and in the maintenance of immune tolerance by the inactivating of self-acting na&#239;ve T cells14,15. The CP plays many important roles in the adaptive immune system, however the molecular mechanisms of CP have yet to be described in detail. Previous studies of CP revealed that exogenous antigens were localized both in the ER and the endosome and were processed by ERAD, suggesting that exogenous antigens are transported from the endosome to the ER for ERAD-like processing and peptide loading16. However, accumulating evidence indicates that the peptide loading of CP is carried out not in the ER but rather in non-classical endocytic compartments, which also have distinctive features of the ER (Figure 1)17,18,19,20,21. To avoid degradation of the antigenic peptide precursors by the high activity of aminopeptidase22 in the cytosol, processing and peptide loading in CP occurs in the proximal area of these non-classical endocytic compartments (Figure 1). Though the characterizations of these endocytic compartments are controversial, there are no existing specific molecules other than exogenous antigens in this compartment.
ERAD is a cellular pathway, which specifically removes misfolded proteins from the ER. In the ERAD pathway, misfolded proteins are retrogradely transported through the ER membrane to the cytoplasm and processed by the ubiquitin-proteasome system23,24,25. When large molecules, such as proteins, are transported through the lipid bilayer, these molecules pass through a molecular apparatus called a translocon, such as the Sec61 complex and Derlin complex in the ER26, and the Tom complex and Tim complex in the mitochondria27. When exogenously-added antigens are transported through the ER membrane, they must penetrate the lipid bilayer in complex with translocons, such as the Sec61 complex. The method described here purified the targeted vesicle by utilizing these membrane-penetrating molecules as markers for the endocytic compartments.
The method described here is a new vesicle purification protocol using the DC-like cell line DC2.428 and biotinylated ovalbumin (bOVA) as an exogenous antigen. The endocytic compartments were purified by streptavidin (SA)-magnetic beads using the membrane-penetrating bOVA as a maker. In this purified microsome, some exogenously added bOVA was still preserved in membrane fractions but were transported to the outside of microsome, and then ubiquitinated and processed in vitro29. This purified microsome contained not only endocytic compartment-specific proteins but also ER-resident proteins for ERAD and the peptide loading complex; suggesting that the cellular compartment is the prospective endocytic compartment for CP29. This protocol is not dependent on the kind of exogenous antigens, and is also applicable for other DC subsets and other cell types, such as macrophages, B cells, and endothelial cells, to clarify the precise molecular mechanism of DCs for proficient CP.

<table border="0" cellpadding="0" cellspacing="0" width="871">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RPMI 1640</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11875-093</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum</td>
			<td style="width:239px;">Equitech bio</td>
			<td style="width:119px;">SFB30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sodium pyruvate</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11360-070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:272px;">MEM non-essential amino acids</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">HEPES</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">2-mercaptoethanol</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">25030-164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penisicillin-Sreptomycin</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:239px;">gibco by life technologies</td>
			<td style="width:119px;">12100-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OVA</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Biotin-protein labelling kit</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F6347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">MG-132&#160;</td>
			<td style="width:239px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">201270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">lactacystin&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">L6785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Dounce homogenizer</td>
			<td style="width:239px;">IUCHI</td>
			<td style="width:119px;">131703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">protease inhibitor cocktails&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">iodixanol&#160;</td>
			<td style="width:239px;">Cosmo bio</td>
			<td style="width:119px;">1114542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">SA-magnetic beads&#160;</td>
			<td style="width:239px;">New England Biolabs</td>
			<td style="width:119px;">201270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">control magnetic beads</td>
			<td style="width:239px;">Chemagen</td>
			<td style="width:119px;">M-PVA012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">magnetic stand</td>
			<td style="width:239px;">BD Biosciences</td>
			<td style="width:119px;">552311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">BCA protein assay kit</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">silver staining kits</td>
			<td style="width:239px;">Cosmo bio</td>
			<td style="width:119px;">423413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Reticulocyte Lysate</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">1730714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Flag-tagged ubiquitin&#160;</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">U5382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-ovalbumin (OVA,mouse)</td>
			<td style="width:239px;">Antibody Shop</td>
			<td style="width:119px;">HYB 094-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">ant-multi-ubiquitin (mouse)</td>
			<td style="width:239px;">MBL</td>
			<td style="width:119px;">D058&#8722;3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">anti-Flag (mouse)</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">trypsin</td>
			<td style="width:239px;">SIGMA</td>
			<td style="width:119px;">85450C</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

To elucidate the molecular mechanism of CP, it is necessary to identify the cellular compartments, where exogenous antigens undergo ERAD-like transport and processing. While observations by immunofluorescent microscopy or by electron microscopy identified the cellular compartment where exogenous antigens accumulated16,17,18,19,...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

The overall goal of this experiment is to purify the membranous compartment, where ERAD-like degradation of exogenous antigens is carried out in cross-presentation.This method can help answer key questions in the immunity period, such as the elucidation of cellular compartment or ERAD-like degradation in cross-presentation.The main advantage of this technique is that the purified vesicles contain all molecular operations.That enables ERAD-like degradation of all exogenous antigens.So this method can provide insight into cross-presentation.By dendritic cells, it can also be applied to other types of antigen-presenting cells which show cross-presentation ability.Generally, individuals new to this method will struggle because it is impossible to exclude all non-specific background.A day before purification, split the DC2.4 cells to 0.1 million cells per mL in a tissue culture plate in RPMI buffer.Avoid keeping the cells at a confluent state to increase probability of exogenous antigen incorporation.Before the DC2.4 cells are semi-confluent, add biotinylated ovalbumin, or bOVA, as an exogenous antigen.Then, incubate for two to four hours.Harvest the cells by gentle pipetting from the tissue plate into a new 50 mL conical tube.Centrifuge the cells at 1000 X G for five minutes at 4

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JoVE Journal

Immunology and Infection

8.1K Views.  Takasaki University of Health and Welfare. The overall goal of this experiment is to purify the membranous compartment, where ERAD-like degradation of exogenous antigens is carried out in cross-presentation.This method can help answer key questions in the immunity period, such as the elucidation of cellular compartment or ERAD-like degradation in cross-presentation.The main advantage of this technique is that the purified vesicles contain all molecular operations.That enables ERAD-like degradation of all exogenous antigens.So this met...