I would like to thank Bianca Moth&eacute;, Carla Oseroff, and Marie-France DelGuercio for introducing me to immunological assays and mouse handling. Work in the lab of G. A. Splitter is funded by NIH grant 1-RO1-AI-073558, GLRCE grant 1-U54-AI-057153, and BARD US-3829-06.

1. Eliciting Target-Specific Effector CTLs by Immunization (Day 0)
<ol>
<li> Before beginning, decide upon a specific effector:target system. In this example of a classical in vivo CTL assay, we will use a purified peptide immunization to elicit specific CD8 + T cells. The same peptide will be used to pulse the syngeneic target cells, creating a specific effector:target system. Alternatively, you may choose to use whole pathogen or protein to obtain your specific effectors and targets.</li>
<li> Decontaminate the work surface inside of a biosafety hood to ensure sterility of the injections. Prepare the peptide immunization by first placing 50 &mu;g of purified peptide into 100 &mu;L 10% DMSO in PBS/mouse contained in a 2 mL tube. Next, add an equal amount of very cold Incomplete Freund's Adjuvant and up to the .2 mL mark with 1 mm beads for mixing. From this point, keep immunogen on ice until injection. Secure lid with Parafilm. </li>
<li> Use a bead beater set to maximum RPMs to mix the emulsion for 3 minutes, promptly placing the emulsion back on the ice after mixing. The resulting emulsion should be similar in consistency to mayonnaise. Allow emulsion to sit at 4&deg; C overnight if time permits (up to a week), mixing further if any separation is noted.</li>
<li> Use a 1 mL syringe and 20 or 20.5 gauge needle to pull up emulsion being careful to not pull up any beads (beads should be larger than gauge of needle, but still be aware).</li>
<li> Secure mouse for subcutaneous injection at the base of the tail. We use a modified plastic 50 mL conical tube to safely and gently immobilize the mouse for injection. Inject the mouse with .2 mL of emulsion. </li>
</ol>
2. Harvesting Target Cells (Day 8)
<ol>
<li> You will need na&iacute;ve syngeneic mice for your target cells. Euthanize na&iacute;ve mice according to AAALAC standards. </li>
<li> Disinfect mouse by spraying 70% Ethanol to minimize the chance of contamination from mouse hair or other external contaminants.</li>
<li> Using dissection scissors make small snip through the skin on the left side of the mouse. Pull skin flap over to uncover peritoneal sac and internal organs.</li>
<li> Snip through the peritoneum, being careful not to compromise any of the underlying organs. The spleen is close to the surface and is similar in color to the liver (dark red) but smaller and more kidney bean in shape. Infected mice generally show notably larger spleens than uninfected controls. </li>
<li> Carefully pull out the spleen, taking care to be gentle so as not to break the spleen into multiple pieces in order to recover maximum cell numbers. Immediately place the spleen into a 15 mL conical containing 10 mLs of prepared media on ice. Continue with remaining mice.</li>
<li> Pour contents of the 15 mL conical tube (media and spleen) into a glass tissue grinder. Grind the spleen using plenty of upper arm strength. When all tissue has lost its color you may assume that you have recovered all cells out of the connective tissue.</li>
<li> Pour contents of the grinder through a 70 &mu;m cell filter into a 50 mL conical tube. Rinse grinder with fresh media and pour through a cell strainer.</li>
<li> Centrifuge at 4&deg; C, 1300 RPM for 7 minutes. Pour off supernatant.</li>
<li> Resuspend in 10 mLs of prepared lysing reagent and incubate at 37&deg; for 7 minutes.</li>
<li> Add up to 50 mLs with fresh media and centrifuge, same as above.</li>
<li> Resuspend in 50 mLs fresh media and centrifuge, same as above (wash step). *Note: At this time you can perform a magnetic cell separation for a specific cell type (i.e. macrophages) if a pure target cell population is desired for transfer into recipient mice. Also, at this time you could introduce an experimental treatment into the culture in order to study the affects of that treatment on cell specific killing.</li>
<li> Count cells and plate 1 x 107 per well in 100&mu;L in a 96 well round bottom tissue culture treated plate. *This is a high concentration of cells/well, but because the assay is a quick one, we have seen no loss in viability of the cells at this concentration.</li>
</ol>
3. Treatment and Labeling of Target Cells
<ol>
<li> Pulse appropriate cells and replicates with pathogen specific peptide or irrelevant peptide control: Add 1 &mu;g/mL peptide. Incubate at 4&deg; C for 1.5 hours. Move to 37&deg; C incubator for last 30 minutes. During this time prepare CFSE reagent.</li>
<li> Prepare CFSE according to manufacturer specifications and dilute to a working concentration between 0.5 and 25 &mu;M. Make "low" concentration 10x lower than "high" concentration CFSE. For example, for [low] add 0.5 &mu;L CFSE to 10 mLs PBS and for [high], add 5 &mu;L CFSE to 10 mLs PBS. Warm prepared CFSE to 37&deg; C before labeling.</li>
<li> Remove plate of cells from incubator and pellet in a high speed centrifuge at room temperature, 1300 RPM for 7 minutes. Flick to remove supernatant.</li>
<li> Resuspend cells in either [low] or [high] CFSE by adding 200 &mu;L of appropriate reagent to the corresponding wells and incubate for 15 minutes at 37&deg; C. </li>
<li> Remove plate of cells from incubator and again pellet at room temperature, 1300 RPM for 7 minutes. Flick to remove supernatant.</li>
<li> Resuspend cells in 200 &mu;L/well of warmed fresh media and incubate for 30 minutes at 37&deg;C.</li>
<li> Remove plate of cells from incubator and pellet at room temperature, 1300 RPM for 7 minutes. Flick to remove supernatant.</li>
<li> Resuspend cells in 100 &mu;L/well PBS.</li>
<li> For adoptive transfer, add one well [low] labeled cells to one well [high] labeled cells per mouse. Resulting injection should be 50% specific targets and 50% irrelevant cells in .2 mLs.</li>
<li> Don't forget to set aside some labeled cells to act as your non-transferred controls during data analysis and for running on the flow cytometer (See Figure 1).</li>
</ol>
4. Adoptive transfer
<ol>
<li> Anesthetize previously peptide immunized mice (From part #1 of this protocol) according to AAALAC standards.</li>
<li> Inject .2 mLs of labeled target cells via retro-orbital route into recipient mice.</li>
<li> Wait 6 hours or other predetermined optimal time before euthanizing the recipient mice.</li>
</ol>
5. Harvesting Labeled Cells
*Note: Steps 5.1 through 5.11 are the same as steps 2.1 through 2.11
<ol start="12">
<li> Count cells and plate out at 1 x 106 per well in 100 &mu;L into 96 well round bottom tissue culture treated plate.</li>
<li> Fix recovered and non-transferred cells by adding 100 &mu;L 2% paraformaldehyde to a final concentration of 1% paraformaldehyde to all wells.</li>
</ol>
6. Flow Cytometry and Data Analysis
<ol>
<li> Run samples on a flow cytometer and analyze the data.</li>
</ol>
7. Representative Results
A diminishing epitope specific peak on the resulting histogram is an indicator of cell specific killing (Figure 1). Use the following equations (applied in Table 1) to determine the actual value of cell specific lysis: 
Ratio = Irrelevant Percentage : Epitope Specific Percentage ( [low] peak:[high] peak), 
Percent Specific Lysis = [1-(Non-transferred control ratio/Experimental ratio)] x 100
<img src="/files/ftp_upload/2250/2250fig1.jpg" alt="Figure 1" /> 
Figure 1. CFSE profile of recovered BALB/c mouse splenocytes. Numbers indicate the percentage of cells of the high or low CFSE phenotype. A) Non-transferred control. All [high] CFSE peaks represent epitope specific target cells, while [low] CFSE peaks represent targets pulsed with irrelevant peptide. B) CFSE labeled cells recovered from PBS immunized recipient mouse (Control). C) CFSE labeled cells recovered from two different peptide specific recipient mice, note the diminished epitope specific peaks.
<img src="/files/ftp_upload/2250/2250fig2.jpg" alt="Figure 2" /> 
Figure 2. Graphical representation of analyzed data. Three data points shown for each group of mice, PBS, Peptide A, and Peptide B.
<table border="1">
<tbody>
 <tr>
 <td>Sample</td>
 <td>CFSE HIGH % Epitope Pulsed</td>
 <td>CFSE LOW % Irrelevant</td>
 <td>Ratio Low:High</td>
 <td>Percent Epitope Specific Killing</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>42.8</td>
 <td>57.2</td>
 <td>1.34</td>
 <td>16.97</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>43.1</td>
 <td>56.9</td>
 <td>1.32</td>
 <td>15.94</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>44.3</td>
 <td>55.7</td>
 <td>1.26</td>
 <td>11.74</td>
 </tr>
 <tr>
 <td>Peptide A</td>
 <td>39.7</td>
 <td>60.3</td>
 <td>1.52</td>
 <td>32.56</td>
 </tr>
 <tr>
 <td>Peptide A</td>
 <td>38.6</td>
 <td>61.4</td>
 <td>1.59</td>
 <td>35.61</td>
 </tr>
 <tr>
 <td>Peptide A</td>
 <td>40.6</td>
 <td>59.4</td>
 <td>1.46</td>
 <td>29.99</td>
 </tr>
 <tr>
 <td>Peptide B</td>
 <td>38.7</td>
 <td>61.3</td>
 <td>1.58</td>
 <td>24.68</td>
 </tr>
 <tr>
 <td>Peptide B</td>
 <td>38.5</td>
 <td>61.5</td>
 <td>1.60</td>
 <td>25.32</td>
 </tr>
 <tr>
 <td>Peptide B</td>
 <td>39.3</td>
 <td>60.7</td>
 <td>1.54</td>
 <td>22.76</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS NT Control</td>
 <td>47.4</td>
 <td>52.6</td>
 <td>1.11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pep A NT Control</td>
 <td>49.4</td>
 <td>50.6</td>
 <td>1.02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pep B NT Control</td>
 <td>45.6</td>
 <td>54.4</td>
 <td>1.19</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>
Table 1. Analysis of representative data. "NT" is non-transferred.

<ol>
	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <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+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

No conflicts of interest declared. Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at The University of Wisconsin-Madison.

This assay can be modified to investigate the proliferative capacity of cells, including clonal expansion, because CFSE divides relatively equally among progeny10,11. It is also possible to use CFSE labeling to investigate cell migration6,12, though there are other cell tracing methods that may be more appropriate depending on your hypothesis, for example tetramers and bioluminescence13, which also may be combined with CFSE labeling.
It is important to note th...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 mm glass beads</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>11079110</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>96-well plate</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3799</td>
<td>&nbsp;</td>
<tr>
<td>Adjulite Incomplete Freund's Adjuvant</td>
<td>&nbsp;</td>
<td>Pacific Immunology</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-5879</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>16000-077</td>
<td>&nbsp;</td>
<tr>
<td>FC 500 Flow Cytometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>Kontes glass tissue grinder</td>
<td>&nbsp;</td>
<td>Kontes</td>
<td>885300-0015</td>
<td>&nbsp;</td>
<tr>
<td>Mini Beadbeater</td>
<td>&nbsp;</td>
<td>Biospec Products</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>305175</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm "M"</td>
<td>&nbsp;</td>
<td>Pechiney Plastic Packaging</td>
<td>PM992</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>157-8</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>PharmLyse lysing reagent</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>555899</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11875-093</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>Vybrant CFSE Kit</td>
<td>&nbsp;</td>
<td>Invitrogen (Molecular Probes)</td>
<td>V12883</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

antigen specific in vivo killing assay using cfse labeled target cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

antigen-specific-in-vivo-killing-assay-using-cfse-labeled-target-cells

Research

JoVE Journal

Immunology and Infection

19.5K 次观看.  University of Wisconsin-Madison. 许多感染引起强烈的CTL反应，但偶尔，响应细胞的数量不关联控制病原体 1。 CTL的质量的措施之一是他们杀了专门的能力 2。 CFSE标签的靶细胞，可用于研究这种CTL反应质量在体内

视频: Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...