The authors thank S. Prettin for expert technical assistance. This work was supported by a grant of the Helmholtz Association of German Research Centers (HGF) that was provided as part of the Helmholtz Alliance &#39;&#39;Immunotherapy of Cancers&#34; (HCC_WP2b).

<ol>
	<li>Amon, L., Hatscher, L., Heger, L., Dudziak, D., Lehmann, C. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+complete+repertoire+of+conventional+dendritic+cell+functions+for+cancer+immunotherapy.">Harnessing the complete repertoire of conventional dendritic cell functions for cancer immunotherapy.</a> Pharmaceutics. 12 (7), 12070663 (2020).</li><li>Banchereau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Wculek, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+in+cancer+immunology+and+immunotherapy.">Dendritic cells in cancer immunology and immunotherapy.</a> Nature Reviews: Immunology. 20 (1), 7-24 (2020).</li><li>Kastenmuller, W., Kastenmuller, K., Kurts, C., Seder, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell-targeted+vaccines--hope+or+hype.">Dendritic cell-targeted vaccines--hope or hype.</a> Nature Reviews: Immunology. 14 (10), 705-711 (2014).</li><li>Bonifaz, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+targeting+of+antigens+to+maturing+dendritic+cells+via+the+DEC-205+receptor+improves+T+cell+vaccination.">In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.</a> Journal of Experimental Medicine. 199 (6), 815-824 (2004).</li><li>Boscardin, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+targeting+to+dendritic+cells+elicits+long-lived+T+cell+help+for+antibody+responses.">Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses.</a> Journal of Experimental Medicine. 203 (3), 599-606 (2006).</li><li>Hossain, M. K., Wall, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Dendritic+Cell+Receptors+as+Targets+for+Enhancing+Anti-Cancer+Immune+Responses.">Use of Dendritic Cell Receptors as Targets for Enhancing Anti-Cancer Immune Responses.</a> Cancers. 11 (3), 11030418 (2019).</li><li>Johnson, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+melanoma+growth+by+targeting+of+antigen+to+dendritic+cells+via+an+anti-DEC-205+single-chain+fragment+variable+molecule.">Inhibition of melanoma growth by targeting of antigen to dendritic cells via an anti-DEC-205 single-chain fragment variable molecule.</a> Clinical Cancer Research. 14 (24), 8169-8177 (2008).</li><li>Trumpfheller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbial+mimic+poly+IC+induces+durable+and+protective+CD4++T+cell+immunity+together+with+a+dendritic+cell+targeted+vaccine.">The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2574-2579 (2008).</li><li>Idoyaga, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparable+T+helper+1+(Th1)+and+CD8+T-cell+immunity+by+targeting+HIV+gag+p24+to+CD8+dendritic+cells+within+antibodies+to+Langerin,+DEC205,+and+Clec9A.">Comparable T helper 1 (Th1) and CD8 T-cell immunity by targeting HIV gag p24 to CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (6), 2384-2389 (2011).</li><li>Sancho, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+therapy+in+mice+via+antigen+targeting+to+a+novel,+DC-restricted+C-type+lectin.">Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin.</a> Journal of Clinical Investigation. 118 (6), 2098-2110 (2008).</li><li>Volckmar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+antigen+delivery+to+dendritic+cells+elicits+robust+antiviral+T+cell-mediated+immunity+in+the+liver.">Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.</a> Scientific Reports. 7, 43985 (2017).</li><li>Kraal, G., Breel, M., Janse, M., Bruin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans'+cells,+veiled+cells,+and+interdigitating+cells+in+the+mouse+recognized+by+a+monoclonal+antibody.">Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.</a> Journal of Experimental Medicine. 163 (4), 981-997 (1986).</li><li>Volckmar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STING+activator+c-di-AMP+exerts+superior+adjuvant+properties+than+the+formulation+poly(I:C)/CpG+after+subcutaneous+vaccination+with+soluble+protein+antigen+or+DEC-205-mediated+antigen+targeting+to+dendritic+cells.">The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.</a> Vaccine. 37 (35), 4963-4974 (2019).</li></ol>

The authors have nothing to disclose.

Chemical conjugation of an endocytosis receptor-specific antibody and a protein antigen provides an efficient and, importantly, also flexible approach for in vivo DC targeting in pre-clinical mouse models. With our protocol we provide an efficient approach for the successful conjugation of the model antigen OVA to a DEC-205-specific IgG antibody.
In our protocol, &#945;DEC-205 is purified from a hybridoma cellline and in the past, we have purified the antibody using protein G sepharos...

Dendritic cells (DC) are central players of the immune system. They are a diverse group of cells specialized in antigen-presentation and their major function is to bridge innate and adaptive immunity1,2. Importantly, DC not only play an important role in efficient and specific pathogen-directed responses but are also involved in many aspects of antitumor immunity1,3.
Due to their exclusive role in host immunity, DC came into focus as target cells for vaccination4. One approach is to target antigens to DC in vivo to induce antigen-specific immune responses and over the last years, a large number of studies have been dedicated to defining suitable receptors and targeting strategies1,4. One example is the C-type lectin receptor DEC-205, which can be targeted by DEC-205-specific antibodies to induce endocytosis. Importantly, DEC-205 targeting in the combination with suitable adjuvants has been shown to efficiently induce long-lived and protective CD4+ and CD8+ T cells, as well as antibody responses, also against tumor antigens3,5,6,7,8,9.
There are a number of studies showing conjugated antigens targeted to DC to be superior to free un-conjugated antigen3,5,10,11,12. This makes the conjugation of the antigen to the respective DC targeting moiety a central step in DC targeting approaches. In the case of DC targeting via antibodies or antibody fragments, antigens can be either chemically or genetically linked and either strategy provides its own (dis)advantages1. On the one hand, in genetically engineered antibody-antigen constructs there is a control over the antigen dose as well as the location providing superior comparability between lots1. At the same time however, chemical conjugation needs less preparation and provides more flexibility especially when attempting to test and compare different antigens and/or vaccination strategies in experimental and pre-clinical models.
Here, we present a protocol for the efficient and reliable chemical conjugation of ovalbumin (OVA) as a model protein antigen to a DEC-205-specific IgG2a antibody (&#945;DEC-205) suitable for in vivo DC targeting in mice. First, &#945;DEC-205 is purified from&#160;NLDC-145 hybridoma cells13. For chemical conjugation, the heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC), which contains NHS (N-hydroxysuccinimide) ester and maleimide groups, is used, allowing covalent conjugation of amine- and sulfhydryl-containing molecules. Specifically, the primary amines of the antibody initially react with sulfo-SMCC and the resulting maleimide-activated &#945;DEC-205 then reacts with the sulfhydryl-containing OVA protein reduced through TCEP-HCl (Tris(2-carboxyethyl) phosphine hydrochloride). The final product is chemically conjugated &#945;DEC-205/OVA (Figure 1). Beyond chemical conjugation itself, our protocol describes removal of excess OVA from the conjugate&#160;as well as the verification of successful conjugation through western blot analysis and a specific enzyme-linked immunosorbent assay. We have successfully employed this approach in the past to chemically conjugate OVA and other proteins or immunogenic peptides to &#945;DEC-205. We demonstrate efficient binding to CD11c+ cells in vitro as well as the efficient induction of cellular and humoral immunity in vivo.
Certainly, there are drawbacks to this method such as in lot-to-lot comparability and in the exact dosing of the antigen within the final conjugate. Nevertheless, chemical conjugation provides experimental flexibility in the choice of the antibody and the protein antigen as compared to genetically engineered constructs. Therefore, we believe this approach is especially valuable in evaluating different antigens for their efficiency in DC targeting in pre-clinical mouse models, importantly also in the context of specific antitumor immune responses.

<table><tbody><tr><td>antibody buffer 2 %</td><td></td><td></td><td>2 % (w/v) Slim-Fast Chocolate powder in TBS-T</td></tr><tr><td>antibody buffer 5 %</td><td></td><td></td><td>5 % milk powder (w/v) in TBS-T</td></tr><tr><td>blocking buffer (ELISA)</td><td></td><td></td><td>10 % FBS in PBS</td></tr><tr><td>blocking buffer 4 %</td><td></td><td></td><td>4 % (w/v) Slim-Fast Chocolate powder in TBS-T</td></tr><tr><td>blocking buffer 10 %</td><td></td><td></td><td>10 % milk powder (w/v) in TBS-T</td></tr><tr><td>cell culture flask T25</td><td>Greiner Bio-One</td><td>690175</td><td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (690195 )</td></tr><tr><td>cell culture flask T75</td><td>Greiner Bio-One</td><td>658175</td><td>we use standard CELLSTAR&amp;nbsp; filter cap cell culture flasks; alternatively use suspension culture flask (658195)&amp;nbsp;</td></tr><tr><td>cell culture flask T175</td><td>Greiner Bio-One</td><td>661175</td><td>we use standard CELLSTAR filter cap cell culture flasks; alternatively use suspension culture flask (661195)</td></tr><tr><td>centrifugal concentrator MWCO 10 kDa</td><td>Sartorius</td><td>VS2001</td><td>Vivaspin 20 centrifugal concentrator</td></tr><tr><td>centrifugal protein concentrator MWCO 100 kDa, 5 - 20 ml</td><td>Thermo Fisher Scientific</td><td>88532</td><td>Pierce Protein Concentrator, PES 5 -20 ml; we use the Pierce Concentrator 150K MWCO 20mL (catalog number 89921), which is however no longer available&amp;nbsp;</td></tr><tr><td>centrifuge bottles</td><td>Nalgene</td><td>525-2314</td><td>PPCO (polypropylene copolymer) with PP (polypropylene) screw closure, 500 ml; obtained from VWR, Germany</td></tr><tr><td>coating buffer (ELISA)</td><td></td><td></td><td>0.1 M sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;) in H&lt;sub&gt;2&lt;/sub&gt;O (pH 9.6)</td></tr><tr><td>desalting columns MWCO 7 kDa</td><td>Thermo Fisher Scientific</td><td>89891</td><td>Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 5 mL</td></tr><tr><td>detection reagent ELISA (HRPO substrate)</td><td>Sigma-Aldrich/Merck</td><td>T8665-100ML</td><td>3,3&amp;prime;,5,5&amp;prime;-Tetramethylbenzidine (TMB) liquid substrate system</td></tr><tr><td>detection reagent western blot (HRPO substrate)</td><td>Roche/Merck</td><td>12 015 200 01</td><td>Lumi-Light Western Blotting Substrate (Roche)</td></tr><tr><td>dialysis tubing MWCO 12 - 14 kDa</td><td>SERVA Electrophoresis</td><td>44110</td><td>Visking dialysis tubing, 16 mm diameter</td></tr><tr><td>ELISA 96-well plate</td><td>Thermo Fisher Scientific</td><td>442404</td><td>MaxiSorp Nunc-Immuno Plate</td></tr><tr><td>fetal calf serum</td><td>PAN-BIOtech</td><td>P40-47500</td><td>FBS Good forte</td></tr><tr><td>ISF-1 medium</td><td>Biochrom/bioswisstec</td><td>F 9061-01</td><td></td></tr><tr><td>milk powder</td><td>Carl Roth</td><td>T145.2</td><td>powdered milk, blotting grade, low in fat; alternatively we have also used conventional skimmed milk powder from the supermarket</td></tr><tr><td>NLDC-145 hybridoma</td><td>ATCC</td><td>HB-290</td><td>if not already at hand, the hybridoma cells can be acquired from ATCC</td></tr><tr><td>non-reducing SDS sample buffer 4 x&amp;nbsp;</td><td></td><td></td><td>for 12 ml: 4 ml of 10 % SDS, 600 &amp;micro;l 0.5 M Tris-HCl (ph 6.8), 3.3 ml sterile H&lt;sub&gt;2&lt;/sub&gt;O, 4 ml glycerine, 100 &amp;micro;l of 5 % Bromphenol Blue</td></tr><tr><td>ovalbumin</td><td>Hyglos (via BioVendor)</td><td>321000</td><td>EndoGrade OVA ultrapure with &amp;lt;0.1 EU/mg</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Gibco Penicillin/Streptomycin 10.000 U/ml; alternatively Gibco Penicillin/Streptomycin 5.000 U/ml (15070-063) can be used</td></tr><tr><td>PETG polyethylene terephthalate glycol cell culture roller bottles</td><td>Nunc In Vitro</td><td>734-2394</td><td>standard PDL-coated, vented (1.2X), 1050 cm&amp;sup2;, 100 - 500 ml volume; obtained from VWR, Germany&amp;nbsp;&amp;nbsp;</td></tr><tr><td>pH indicator strips</td><td>Merck</td><td>109535</td><td>pH indicator strips 0-14</td></tr><tr><td>polyclonal goat &amp;alpha;rat-IgG(H+L)-HRPO (western blot)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>112-035-062</td><td>obtained from Dianova, Germany; used at 1:5000 for western blot</td></tr><tr><td>polyclonal goat &amp;alpha;rat-IgG+IgM-HRPO antibody&amp;nbsp; (ELISA)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>112-035-068</td><td>obtained from Dianova, Germany; used at 1:2000 for ELISA</td></tr><tr><td>polyclonal goat &amp;alpha;rabbit-IgG-HRPO (western blot)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>111-035-045&amp;nbsp;</td><td>obtained from Dianova, Germany; used at 1:2000 for western blot</td></tr><tr><td>polyclonal rabbit &amp;alpha;OVA (ELISA)</td><td>Abcam</td><td>ab181688</td><td>used at 3 ng/&amp;micro;l</td></tr><tr><td>polyclonal rabbit &amp;alpha;OVA antibody (western blot)</td><td>OriGene</td><td>R1101</td><td>used at 1:3,000 for western blot</td></tr><tr><td>Protein G Sepharose column</td><td>Merck/Millipore</td><td>P3296</td><td>5 ml Protein G Sepharose, Fast Flow are packed onto an empty column PD-10 (Merck, GE 17-0435-01)</td></tr><tr><td>protein standard</td><td>Thermo Fisher Scientific</td><td>26616</td><td>PageRuler Prestained Protein ladder 10 - 180 kDa</td></tr><tr><td>PVDF (polyvinylidene difluoride) membrane</td><td>Merck/Millipore</td><td>IPVH00010</td><td>immobilon-P PVDF (polyvinylidene difluoride) membrane</td></tr><tr><td>rubber plug</td><td>Omnilab</td><td>5230217</td><td>DEUTSCH &amp;amp; NEUMANN rubber stoppers (lower &amp;Phi; 17 mm; upper &amp;Phi; 22 mm)</td></tr><tr><td>silicone tube</td><td>Omnilab</td><td>5430925</td><td>DEUTSCH &amp;amp; NEUMANN (inside &amp;Phi; 1 mm; outer &amp;Phi; 3 mm)</td></tr><tr><td>Slim-Fast</td><td></td><td></td><td>we have used regular Slim-Fast Chocolate freely available at the pharmacy as in this western blot approach it yielded better results than milk powder</td></tr><tr><td>stopping solution (ELISA)</td><td></td><td></td><td>1M H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>sulfo-SMCC</td><td>Thermo Fisher Scientific</td><td>22322</td><td>Pierce Sulfo-SMCC Cross-Linker; alternatively use catalog number A39268 (10 x 2 mg)</td></tr><tr><td>syringe filter unit 0.22 &amp;micro;m&amp;nbsp;</td><td>Merck/Millipore</td><td>SLGV033RS</td><td>Millex-GV Syringe Filter Unit, 0.22 &amp;micro;m, PVDF, 33 mm, gamma sterilized&amp;nbsp;</td></tr><tr><td>syringe 10 ml</td><td>Omnilab</td><td></td><td>Disposable syringes Injekt&amp;reg; Solo B.Braun</td></tr><tr><td>Sterican&amp;reg; cannulas</td><td>B. Braun</td><td></td><td>Sterican&amp;reg; G 20 x 1 1/2&quot;&quot;; 0.90 x 40 mm; yellow</td></tr><tr><td>TBS-T</td><td></td><td></td><td>Tris-buffered saline containing 0.1 % (v/v) Tween 20</td></tr><tr><td>TCEP-HCl</td><td>Thermo Fisher Scientific</td><td>A35349</td><td></td></tr><tr><td>tubing connector</td><td>Omnilab</td><td></td><td>Kleinfeld miniature tubing connectors for silicone tube</td></tr></tbody></table>

chemical conjugation of a purified dec-205-directed antibody with full-length protein for targeting mouse dendritic cells in vitro and in vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical conjugation of &#945;DEC-205 to OVA protein using this protocol will typically allow efficient generation of &#945;DEC-205/OVA for in vivo DC targeting approaches. There are different strategies to verify the technique itself and to test the functionality of the yielded conjugate. Western blot analysis and ELISA are used to verify successful conjugation and at the same time detect potentially left free OVA (Figure 2). In vitro bindi...

Watch this Scientific Journal Video about Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo at JoVE.com

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

chemical-conjugation-purified-dec-205-directed-antibody-with-full

Research

JoVE Journal

Cancer Research

3.3K Views.  Helmholtz Centre for Infection Research. Dit protocol waarvoor alle experimentele condities zorgvuldig zijn geoptimaliseerd, kan worden gebruikt om een succesvolle en efficiënte chemische conjugatie van het DEC-205-antilichaam tegen OVA-antigeen te vergemakkelijken. De belangrijkste voordelen van deze techniek zijn dat het een flexibele selectie van het eiwitantigeen en antilichaam mogelijk maakt en dat het niet afhankelijk is van genetische fusie van deze componenten. We hebben dit protocol ook gebruikt om conjugaten van hepatitis...

Video: Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Biology

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Immunology and Infection

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Lentiviral vectors (LVs) are a powerful means of delivering genetic material to many types of cells. Because of safety concerns associated with these HIV-1 derived vectors, producing large quantities of LVs is challenging. In this paper, we report a method for producing high titers of self-inactivating LVs. We retrovirally transduce the tet-off stable producer cell line GPR to generate a cell line, GPRS, which can express all the viral components, including a dendritic cell-specific glycoprotein, SVGmu. Then, we use concatemeric DNA transfection to transfect the LV transfer plasmid encoding a reporter gene GFP in combination with a selectable marker. Several of the resulting clones can produce LV at a titer 10-fold greater than what we achieve with transient transfection. Plus, these viruses efficiently transduce dendritic cells in vitro and generate a strong T cell immune response to our reporter antigen. This method may be a good option for producing strong LV-based vaccines for clinical studies of cancer or infectious diseases.

Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Medicine

Enzyme-Linked Immunosorbent Assay to Verify Chemically-Coupled Antibody-Antigen Conjugates

Source: Volckmar, J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62018">Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.</a> J. Vis. Exp. (2021)
This video demonstrates the detection of chemically conjugated antibody-antigen complexes via the sandwich enzyme-linked immunosorbent assay, or ELISA, technique. The captured antibody binds to the specific epitope of the antigen of the antibody-antigen conjugate, which can be later visualized using an enzyme-linked secondary antibody.

Source: Volckmar, J. et al., Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In ...

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Immunoassays

Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells

Source: Wei, H., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57637">Development and Functional Characterization of Murine Tolerogenic Dendritic Cells.</a> J. Vis. Exp. (135), (2018).
This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.

1. Evaluate the Function of TolDCs In Vitro and In Vivo

	T-cell Syngeneic Proliferation Assay
	
		Sterilize all the surgical instruments via autoclaving ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

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