We thank L. Kuhn for technical assistance with tissue processing and tetramer production. This work was funded by the National Institutes of Health (R01 AI107020 and P01 AI165072 to J.J.M., T32 AI007512 to D.S.S.), the Massachusetts Consortium on Pathogen Readiness (J.J.M), and the Massachusetts General Hospital Executive Committee on Research (J.J.M.).

The procedures described in this protocol are approved and developed in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Massachusetts General Hospital, an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal management program. Experiments were performed on 8-12-week-old male and female mice on a C57BL/6 genetic background bred and maintained in the MGH animal facility under specific pathogen-free conditions.
1. Preparing stock solutions
<ol>
	<li>Prepare HEPES buffer by dissolving 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl2, 150 mM NaCl, and 1 mM MgCl2, and adjust to pH 7.4.</li>
	<li>Prepare 10x Liberase stock solution by dissolving 700 &#181;g/mL of Liberase Thermolysin Medium (TM) (see Table of Materials) in Hank&#39;s Balanced Salt Solution with Ca++ and Mg++ (see Table of Materials).</li>
	<li>Prepare 10x aminoguanidine stock solution by dissolving 100 mM Aminoguanidine Hemisulfate Salt (see Table of Materials) in Hank&#39;s Balanced Salt Solution with Ca++ and Mg++ (see Table of Materials).</li>
	<li>Prepare complete Eagle&#39;s Ham&#39;s Amino Acids (EHAA) medium by mixing EHAA medium (see Table of Materials) with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, 50 &#181;g/mL gentamycin, 2 mM L-glutamine, and 55 &#181;M 2-mercaptoethanol. 
	NOTE: Other common T cell media, such as RPMI or DMEM, may also be used.</li>
	<li>Prepare sorter buffer by mixing 1x PBS with 2% FBS and 0.05% sodium azide.</li>
	<li>Prepare Fc block by diluting purified anti-mouse CD16/32 antibody (see Table of Materials) at 1:100 v/v in sorter buffer.</li>
	<li>Prepare Liberase TM/DNase digestion solution by adding 100 &#181;g/mL Liberase TM and 50 &#181;g/mL DNase I (see Table of Materials) in RPMI 1640 medium without L-glutamine (see Table of Materials).</li>
	<li>Prepare ketamine/xylazine mixture by adding 10 mg/mL ketamine (see Table of Materials) and 1 mg/mL xylazine (see Table of Materials) in normal saline (0.9% NaCl) (see Table of Materials).</li>
</ol>
2. Generating single-cell suspension from lung tissue via automated tissue dissociator
<ol>
	<li>For each mouse, prepare 4 mL of ice-cold HEPES buffer in a specialized tissue dissociator tube (see Table of Materials) and keep it on ice.</li>
	<li>Anesthetize mice by intraperitoneal injection of 150-200 &#181;L of ketamine/xylazine mixture. Inject 1-3 &#181;g of fluorophore-conjugated anti-mouse CD45 antibody (see Table of Materials) diluted in 100 &#181;L normal saline intravenously into each anesthetized mouse. Euthanize the mouse 3 min after antibody administration with an intraperitoneal injection of 500-600 &#181;L of ketamine/xylazine mixture.</li>
	<li>Resect the lungs and place them in their respective tissue dissociator tube chilled on ice. Place the tubes onto the automated tissue dissociator (see Table of Materials) and run the first program (preset program) for lung tissue.</li>
	<li>Add 500 &#181;L of Liberase stock solution and 500 &#181;L of aminoguanidine stock solution to each tube containing the dissociated lung tissue. Incubate on a nutating mixer for 30 min at 37 &#176;C.</li>
	<li>Place the tubes back on the dissociator and run the second program (preset program) for lung tissue.</li>
	<li>Pour the single-cell suspension from the tissue dissociator tube through a 100 &#181;m cell strainer (see Table of Materials) into a 50 mL conical tube. Rinse the tissue dissociator tube with 5 mL of complete EHAA (or any equivalent T cell medium) and pass through the strainer into the 50 mL tube.</li>
	<li>Remove the strainer and raise the volume of the cell suspension to 50 mL with complete EHAA. Centrifuge the cell suspension at 400 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant, leaving behind approximately 100 &#181;L of supernatant and cell pellet.</li>
	<li>Add approximately 100 &#181;L of Fc Block solution to bring the total volume up to 200 &#181;L and vortex vigorously to resuspend the pellet.</li>
</ol>
3. Alternative protocol: Generating single-cell suspension from lung tissue via manual dissociation
<ol>
	<li>For each mouse, prepare 1 mL of complete EHAA in a 1.5 mL microfuge tube and keep on ice.</li>
	<li>Anesthetize mice by intraperitoneal injection of 150-200 &#181;L of ketamine/xylazine mixture. Inject 1-3 &#181;g of fluorophore-conjugated anti-mouse CD45 antibody diluted in 100 &#181;L of normal saline intravenously into each anesthetized mouse. Euthanize the mouse 3 min after antibody administration with an intraperitoneal injection of 500-600 &#181;L of ketamine/xylazine mixture.</li>
	<li>Resect the lungs and place them in their respective 1.5 mL microfuge tube chilled on ice.</li>
	<li>Once all the lungs have been harvested, transfer each lung to a fresh microfuge tube without any cell media.</li>
	<li>Cut the lungs into small fragments (~2-3 mm chunks) using scissors within the microfuge tube.</li>
	<li>Add 1 mL of Liberase TM/DNase I digestion cocktail to each tube.</li>
	<li>Incubate for 30 min at 37 &#176;C in a water bath. Return the samples to ice afterward to prevent over-digestion.</li>
	<li>Pour the sample over a 100 &#181;m cell strainer placed on top of a 50 mL conical tube. Mash the tissue against the mesh with the rubber end of the plunger from a sterile 1 mL syringe.</li>
	<li>Rinse the strainer with cold sorter buffer, collecting a final volume of 7 mL in the tube. Centrifuge the cell suspension at 400 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant, leaving approximately 100 &#181;L of supernatant and cell pellet.</li>
	<li>Add 100 &#181;L of Fc Block solution to bring the total volume up to 200 &#181;L and vortex vigorously to resuspend the pellet.</li>
</ol>
4. Enriching the lung sample for T cells via magnetic bead enrichment
<ol>
	<li>Add 50 &#181;L of anti-mouse CD90.2 microbeads (see Table of Materials). Vortex and incubate for 10 min at 4 &#176;C. 
	NOTE: CD90.2 corresponds to the allele expressed by T cells in C57BL/6 mice. Check allele genotype if other strains of mice are used.</li>
	<li>In the meantime, place a paramagnetic cell separation column (see Table of Materials) onto a one or four position cell separation magnet (see Table of Materials). Position an open 15 mL conical tube under each column to capture the flow through. Prime the column with 3 mL of cold sorter buffer, allowing the column to drain by gravity into the conical tube below.</li>
	<li>Once the cells have incubated with the microbeads for 10 min, add cold sorter buffer to a volume of 1 mL and transfer through a 100 &#181;m cell strainer placed atop the column. Collect the column flow through into a fresh 15 mL conical tube placed below the column.</li>
	<li>Once the cell suspension has completely drained through the column by gravity, wash the column by rinsing the original tube with an additional 3 mL of cold sorter buffer and applying it to the column through the mesh, thereby rinsing the strainer. Remove the strainer.</li>
	<li>When the sample has again completely drained into the column, and no further flowthrough is exiting the column, remove the column from the magnet and place it atop a fresh 15 mL conical tube.</li>
	<li>Add 5 mL of cold sorter buffer to the column.</li>
	<li>Immediately elute the cells bound to the column by pushing the column plunger into the top in one continuous motion and forcing the sorter buffer out of the bottom of the column into the new tube.</li>
	<li>Centrifuge the eluted samples at 400 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant, leaving behind approximately 100 &#181;L of supernatant and cell pellet.</li>
	<li>Add&#160;100&#160;&#181;L&#160;of cold sorter&#160;buffer to bring the total volume up to 200&#160;&#181;L&#160;and vortex vigorously to resuspend the pellet.</li>
	<li>Analyze the flowthrough sample containing the unbound cell fraction, if desired.</li>
</ol>
5. Staining the antigen-specific T cells with peptide:MHC tetramers
<ol>
	<li>Add 1 &#181;L of 10 &#181;M Dasatinib (see Table of Materials) to each sample, vortex, and incubate for 5 min at RT.</li>
	<li>Add PE- or APC-conjugated peptide:MHC tetramer to a final concentration of 10 nM (or empirically optimized concentration for a given tetramer).</li>
	<li>Vortex and incubate in the dark for 1 h at RT (or empirically optimized time and temperature).</li>
</ol>
6. Analyzing the tetramer-labeled T cells by flow cytometry
<ol>
	<li>While the sample is being stained with peptide:MHC tetramers, prepare a master mix of antibodies to stain the surface markers of the cells (Table 1). 
	&#8203;NOTE: Avoid fluorophores that conflict with the intravenously administered CD45 antibody or the tetramers.</li>
	<li>With 15 min left in the incubation period for tetramer-staining, add surface antibody master mix at 1:100 (or empirically optimized concentration) to each sample.</li>
	<li>Continue to incubate in the dark at RT for the remainder of the 1 h incubation period.</li>
	<li>Add 50,000 flow cytometry count beads (see Table of Materials) and add cold sorter buffer to a final volume of approximately 5 mL. Centrifuge the stained sample at 400 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant, leaving behind approximately 100 &#181;L of supernatant and cell/bead pellet.</li>
	<li>Resuspend the sample with an additional 200 &#181;L of sorter buffer and transfer the sample to a 5 mL fluorescence activated cell sorting (FACS) tube.</li>
	<li>Analyze the sample on a flow cytometer. Collect as many cells as possible, up to a maximum of 2,500,000 total events. Ensure that at least 20,000 count bead events have been collected. Keep the acquisition rate at or below 5,000 events per second.</li>
	<li>Save all data as FCS files.</li>
</ol>
&#160;&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorochrome</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>BUV395</td>
			<td>CD90.2</td>
		</tr>
		<tr>
			<td>Pacific Blue</td>
			<td>CD45 (added earlier by i.v. injection)</td>
		</tr>
		<tr>
			<td>Pacific Orange</td>
			<td>CD8</td>
		</tr>
		<tr>
			<td>Brilliant Violet 785</td>
			<td>CD4</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>CD3</td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5</td>
			<td>Dump (B220, CD11b, CD11c, F4/80)</td>
		</tr>
		<tr>
			<td>PE</td>
			<td>pMHC tetramer or phenotypic marker</td>
		</tr>
		<tr>
			<td>PE-Cy7</td>
			<td>Phenotypic marker (e.g. CD44, PD-1, CD69, etc.)</td>
		</tr>
		<tr>
			<td>APC</td>
			<td>pMHC tetramer or phenotypic marker</td>
		</tr>
		<tr>
			<td>AlexaFluor 700</td>
			<td>Phenotypic marker (e.g. CD44, PD-1, CD69, etc.)</td>
		</tr>
		<tr>
			<td>APC-Cy7</td>
			<td>Live/dead stain</td>
		</tr>
	</tbody>
</table>
Table 1: Sample staining matrix. A typical panel of fluorophore-conjugated flow cytometry antibodies utilized for identifying and characterizing antigen-specific T cells.
7. Analyzing data
<ol>
	<li>Analyze the FCS data files using appropriate flow cytometry software.
	<ol>
		<li>Set up a sequence of successive inclusion gates to identify 1) lymphocyte-like, 2) single, 3) live, 4) extravascular, 5) dump-negative, and 6) CD90.2-positive events that are either CD4 or CD8 positive (Figure 2).</li>
		<li>Identify the tetramer-positive T cells within these populations.</li>
	</ol>
	</li>
	<li>Determine absolute tetramer-positive cell counts by calculating the ratio of beads added (50,000) to the number of beads collected during the sample run and multiplying the value with the total number of tetramer-positive cells collected. 
	<img alt="Equation 1" src="/files/ftp_upload/66939/66939eq01v2.jpg" style="margin: auto;" /></li>
</ol>

Isolation and Characterization of Antigen-Specific T Cells in Mouse Lungs

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<li>Zhang, Z., Legoux, F. P., Vaughan, S. W., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Opposing+peripheral+fates+of+tissue-restricted+self+antigen-specific+conventional+and+regulatory+CD4%28%2B%29+T+cells%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Opposing peripheral fates of tissue-restricted self antigen-specific conventional and regulatory CD4(+) T cells.</a> Eur J Immunol. 50 (1), 63-72 (2020).</li>
</ol>

The authors have no conflicts to disclose.

Prior characterizations of antigen-specific T cells from the lungs have benefitted from the robust numbers of antigen-specific T cells that expand following an acute priming event such as intranasal immunization or infection20,21,22. However, rarer T cell populations in the lungs, such as self antigen-specific T cells or tissue-resident memory T cells, are difficult to detect without some form of sample enrichment for the T cells of interest15,16,23. We have achieved this goal by combining lung tissue dissociation techniques previously described to yield a high percentage of viable lymphocytes24,25 with a strategy to discriminate intravascular from parenchymal T cells and a general T cell-based enrichment process followed by tetramer staining17. Using this protocol, we have demonstrated the ability to identify and characterize rare T cells that are specific against self antigens in the lung that arise in response to direct immunization with exogenous peptide or indirect activation from antigen release during tissue damage15,16. Several considerations were made in the development of this protocol.
We have described two different lung tissue processing protocols that have worked well. One involves the use of an automated tissue dissociation instrument, while the other relies on a manual process. Importantly, both methods utilize Liberase TM, a blend of collagenase I and II which also contains a metalloprotease thermolysin. This enzyme blend has been demonstrated to increase digestive activity over a single collagenase cocktail and has been demonstrated to work well with the recovery of leukocytes from lung tissue26.
The automated tissue dissociation protocol requires a large upfront cost for the machine and specialized tissue dissociation tubes, but the tubes can be washed and reused along with cell strainers, which are also generally reusable. The manual dissociation protocol does not incur the upfront cost of specialized equipment, but it is labor-intensive and prolongs the harvest as each sample is chopped up individually. There are other differences between the protocols regarding the use of aminoguanidine or DNase I to limit cell death and cell clumping, but we have not carefully compared the impact of these additives. The decision to use either protocol should depend on personal experience in the type of study being performed as well as the availability of an automated tissue dissociation instrument, which would represent the biggest factor in cost. Indeed, elements of the two protocols can be mixed and matched to provide the best performance for the specific needs of the study.
The protocol mentioned above notably excludes the use of a density gradient to isolate lymphocytes from the single-cell suspension prepared from tissue dissociation. We have found that the magnetic T cell enrichment step needed for the identification of rare antigen-specific T cells obviates this step. However, in cases where antigen-specific T cell frequencies are high enough to eliminate the need for a T cell enrichment step altogether, a density gradient is recommended as it will provide a substantial amount of target cell enrichment by removing a large number of nonlymphoid cells. Additionally, if the lung single-cell preparations are particularly large and/or messy, a density gradient step may be used prior to magnetic enrichment to remove excessive cell debris that might later clog the columns (steps 4.4-4.7).
We opted for a general enrichment of all T cells based on the CD90 pan T cell marker (CD90.2 for the allele expressed in C57BL/6 mice) instead of a more direct tetramer-based cell enrichment strategy. This offers several advantages. (1) CD90-based enrichment results in higher numbers of antigen-specific events detected on flow cytometry than either unenriched or tetramer-based enrichment samples (Figure 4). This contributes to better estimates of rare antigen-specific T cell frequencies without necessitating the collection of excessive numbers of total events (&#62;2,500,000), which prolongs sample collection times and worsens signal-to-noise resolution. (2) Unlike tetramer-based enrichment, CD90-based enrichment allows for evaluation of the entire T cell compartment of the lung, including CD4+ and CD8+ T cells, intravascular vs. extravascular T cells, and antigen-specific vs. antigen-non-specific, all in one sample (Figure 2). (3) Because the CD90-based enrichment step occurs prior to tetramer staining, it minimizes the time before tetramer-labeled cells are analyzed on the flow cytometer, thereby limiting the potential loss of signal from tetramer detachment.
The T cell enrichment process in this protocol is based on positive selection with CD90.2 magnetic beads, but similar results can be achieved with negative selection strategies provided by CD4, CD8, or pan T cell isolation kits. We describe the use of a specific brand of microbeads, columns, and magnets, but other magnetic enrichment systems should work, too. Again, decisions should be based on performance in the specific study being performed as well as overall costs, which can be substantial. In our experience with enrichment protocols, there is a general tradeoff between target cell yield and purity, and for the purposes of detecting extremely limited numbers of antigen-specific T cells, yield takes precedence.
While the representative results depict experiments involving CD4+ T cells and peptide:MHC class II tetramers, this protocol is just as effective for CD8+ T cells and peptide:MHC class I tetramers. In such cases, considerations should be made for the potentially different conditions required by class I tetramer staining (concentration, time, temperature). Moreover, we do not see any reasons precluding its use for antigen-specific B cell studies with appropriate fluorochrome-labeled B cell antigen reagents27.
Lastly, the protocol described here includes the addition of the protein kinase inhibitor Dasatinib to the sample prior to tetramer staining. Dasatinib blocks TCR signaling and internalization, which improves tetramer-labeling of T cells while also potentially reducing tetramer-induced T cell death19. We found the benefit of Dasatinib treatment most prominently in the detection of the rarer self antigen-specific population. This is likely due to the fact that self antigen-specific T cells express lower affinity TCR due to the effects of negative selection during central tolerance28,29,30,31.
The protocol used here has been successfully combined with intracellular staining protocols to analyze transcription factor and cytokine expression in rare lung-resident antigen-specific T cells. We have also successfully flow-sorted these rare populations for downstream analyses, including single-cell transcriptomics and TCR sequencing. The continued use and development of this protocol should greatly benefit studies of adaptive immunity in lung pathophysiology.

At the heart of the adaptive immune system lies the ability of a T cell to recognize and respond to a specific antigen. When and where a T cell responds to its cognate antigen determines the balance of infection and autoimmunity, homeostasis and cancer,&#160;health and disease1. It follows that the study of T cells in a specific context of immunity should focus on the cells with specificity for a relevant antigen of interest. Among the technological advancements that have greatly enhanced the ability to characterize antigen-specific T cell populations are fluorescently labeled soluble multimers (usually tetramers) of major histocompatibility complex (MHC) class I or class II molecules complexed to antigenic peptide epitopes, better known as &#34;peptide:MHC tetramers&#34;2,3,4,5. By representing the natural ligands of T cell antigen receptors (TCRs), peptide:MHC class I and class II tetramers provide a means to directly identify antigen-specific CD8+ and CD4+ T cells, respectively, within the endogenous repertoire of T cells in the immune system without a requirement for a response to antigen stimulation in an assay. Tetramers represent a more elegant approach to the study of antigen-specific T cells than TCR transgenic T cell adoptive transfer models6, and have been increasingly used to identify foreign and self antigen-specific T cell populations in both experimental mouse models and human disease4,5.
While tetramers can readily identify high-frequency populations of T cells that have expanded in response to antigen stimulation, their use for naive, self antigen-specific, or memory T cells is limited by the very low frequencies of these populations7. Our group and others have developed and popularized tetramer-based magnetic enrichment strategies that increase the sensitivity of detection to enable studies of these cell populations in mouse lymphoid tissues8,9,10,11.
The emergence of tissue-resident T cells in the field has placed a heightened emphasis on developing new ways to investigate T cells in the nonlymphoid space. Like many other mucosal surfaces, T cells in the lungs encounter a range of self and foreign antigens derived from host epithelium, commensal and infectious microbes, and environmental entities, including allergens. Transcriptional analysis of T cells harvested from nonlymphoid tissue (NLT) demonstrates a memory-like phenotype that bears unique tissue-specific fate and function, often directed at trafficking and tissue homeostasis12. Moreover, tissue-resident memory T cells (Trms) tend to be more clonally restricted than those in circulation13. Determining how and why antigens drive T cell residence in NLT is critical to understanding how the immune system protects against infection, maintains tissue homeostasis, and, at times, devolves into autoimmunity. However, there appears to be greater attrition amongst tissue-resident T cells from the lungs compared to other NLT14. Accordingly, the ability to identify and characterize endogenous T cells of the lung with a given antigen-specificity is limited by their inherent rarity.
By combining the use of magnetic bead-based cell enrichment techniques and peptide:MHC tetramer staining, we have succeeded in detecting expanded but rare self antigen-specific T cells in mouse lungs15,16. Here, we present a detailed description of a protocol that we have optimized to reliably isolate and characterize any rare antigen-specific T cell population present in mouse lungs (Figure 1). This protocol incorporates an in vivo antibody staining step to distinguish tissue-resident from vascular T cells17 followed by two different methods for lung tissue processing to accommodate resource availability. This is then followed by a general T cell magnetic enrichment step, tetramer staining, and analysis by flow cytometry. Cell viability and tetramer staining is further enhanced in this protocol by the addition of aminoguanidine, which blocks inducible nitric oxide synthase (iNOS)-mediated T cell activation-induced apoptosis18 and Dasatinib which limits TCR downregulation19. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology and is highly adaptable for a variety of downstream analyses. Although naive T cells are not likely to be found in the lungs, we believe this protocol will be particularly helpful for the study of self antigen-specific T cells and Trms in the lungs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66939/66939fig01.jpg" /> 
Figure 1: Overview of protocol workflow. Lungs are harvested from mice and dissociated into single cells. Samples are subsequently enriched for T cells prior to staining with peptide:MHC tetramers and fluorescently-labeled antibodies for flow cytometric analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66939/66939fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>100 mm cell strainer</td><td>Fisher Scientific</td><td>22-363-549</td><td></td></tr><tr><td>10x PBS without Ca&lt;sup&gt;++&lt;/sup&gt; or Mg&lt;sup&gt;++&lt;/sup&gt;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>1x PBS without Ca&lt;sup&gt;++&lt;/sup&gt; or Mg&lt;sup&gt;++&lt;/sup&gt;</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>AccuCheck Counting Beads</td><td>Invitrogen</td><td>PCB100</td><td></td></tr><tr><td>Aminoguanidine Hemisulfate Salt</td><td>Sigma-Aldrich</td><td>A7009</td><td></td></tr><tr><td>CD90.2 microbeads, mouse</td><td>Miltenyi</td><td>130-121-278</td><td></td></tr><tr><td>Cell separation magnet (MidiMACS Separator)</td><td>Miltenyi</td><td>130-042-302</td><td>Holds single LS column</td></tr><tr><td>Cell separation magnet (QuadroMACS Separator)</td><td>Miltenyi</td><td>130-090-976</td><td>Holds 4 LS columns</td></tr><tr><td>Dasatinib</td><td>Sigma-Aldrich</td><td>CDS023389</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Eagle&amp;rsquo;s Ham&amp;rsquo;s Amino Acids medium</td><td>Sigma-Aldrich</td><td>C5572</td><td></td></tr><tr><td>gentleMACS</td><td>Miltenyi</td><td>130-093-235</td><td>Automated tissue dissociator</td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi</td><td>130-093-237</td><td>Automated tissue dissociator tubes</td></tr><tr><td>Hank&#039;s Balanced Salt Solution with Ca&lt;sup&gt;++&lt;/sup&gt; or Mg&lt;sup&gt;++&lt;/sup&gt;</td><td>Corning</td><td>21-020-CM</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Ketamine</td><td>Vedco</td><td>NDC 50989-996-06</td><td></td></tr><tr><td>Liberase TM</td><td>Roche</td><td>5401119001</td><td></td></tr><tr><td>Pacific Blue anti-mouse CD45 antibody (Clone: 30-F11)</td><td>Biolegend</td><td>103126</td><td></td></tr><tr><td>Paramagnetic cell separation columns (LS Columns)</td><td>Miltenyi</td><td>130-042-401</td><td>Comes with plunger</td></tr><tr><td>Purified anti mouse CD16/32 antibody (Clone: 93)</td><td>Biolegend</td><td>101302</td><td></td></tr><tr><td>RPMI 1640 medium without L-glutamine</td><td>Corning</td><td>15-040-CM</td><td></td></tr><tr><td>Sodium Chloride 0.9% (Normal Saline)</td><td>Cytiva</td><td>Z1376</td><td></td></tr><tr><td>Xylazine</td><td>Pivetal</td><td>NDC 466066-750-02</td><td></td></tr></tbody></table>

identification of rare antigen-specific t cells from mouse lungs with peptide:major histocompatibility complex tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Figure 2 depicts the representative gating strategy used in the identification of rare antigen-specific CD4+ T cells in the lungs with peptide:MHC class II tetramers. The same process can be applied for antigen-specific CD8+ T cells with peptide:MHC class I tetramers (data not shown).
Due to the high number of nonlymphoid cells in the lungs, the reliable detection of rare antigen-specific T cells by tetramer requires some form of prior enrichment for the T cells of interest to improve signal-to-noise. Figure 3 illustrates the efficacy of the CD90.2 antibody-based magnetic enrichment of total T cells from lung tissue that we have described in this protocol. On average, we recover greater than 99% of all CD90.2+ T cells from the lungs with greater than 90% purity via this enrichment process. Figure 4 illustrates the improved detection of rare tetramer-positive CD4+ T cells resulting from this enrichment versus no enrichment or a direct tetramer-based cell enrichment strategy.
Figure 5 illustrates the improved tetramer-labeling of antigen-specific T cells treated with Dasatinib, particularly in rare populations.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66939/66939fig02.jpg" /> 
Figure 2: Gating strategy for flow cytometry analysis. Sequential inclusion and exclusion gates used to identify rare self antigen-specific CD4+ T cells that accumulate in the lungs following acute tissue injury. Count beads can be identified directly based on their fluorescence along the FITC channel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66939/66939fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66939/66939fig03.jpg" /> 
Figure 3: CD90.2 antibody-based magnetic enrichment of total T cells in the lungs. Representative flow cytometry plots are shown for the unbound (left) and bound (right) fractions of a lung sample following enrichment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66939/66939fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66939/66939fig04.jpg" /> 
Figure 4: Comparison of cell enrichment strategies to aid detection of rare antigen-specific T cells in the lungs. Three mice expressing a model self antigen in lung epithelium were immunized subcutaneously with a peptide epitope from the self antigen emulsified in Complete Freund&#39;s Adjuvant (CFA). After 7 days, mouse lungs were harvested, combined, and split into three equal samples for tetramer-staining following no cell enrichment (left), CD90.2 antibody-based cell enrichment (center), or tetramer-based cell enrichment (right). Samples were analyzed by flow cytometry for no more than 1,500,000 total events (CD4+ gated events shown). Numbers of tetramer-positive events are noted in black, while the computed total numbers of antigen-specific T cells in the lung samples are indicated in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66939/66939fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66939/66939fig05.jpg" /> 
Figure 5: Dasatinib treatment enhances tetramer-staining. Wildtype mice (left) or mice expressing a model self antigen in lung epithelium (right) were immunized subcutaneously with a peptide epitope from the self-antigen emulsified in CFA. The spleens and lymphoid organs from the mice were harvested, combined, and split into two equal samples for labeling with peptide:MHC class II tetramers with or without dasatinib pre-treatment. Histograms of tetramer-staining are overlaid, comparing samples treated with (red) and without (blue) dasatinib. The median fluorescence intensity of each sample is indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66939/66939fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Optimized Protocol for Detecting Antigen-Specific T Cells in Mouse Lungs Using Tetramers

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

identification-rare-antigen-specific-t-cells-from-mouse-lungs-with

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

914 Views.  Massachusetts General Hospital. Ons laboratorium bestudeert de ontwikkeling van CD4 T-cellen in verschillende contexten van tolerantie en immuniteit. In onze projecten zijn we vooral geïnteresseerd in hoe blootstelling aan antigeen de ontwikkeling van fenotypische subsets van T-cellen stimuleert die een immuungeheugen opzetten dat de tolerantie voor zichzelf of de immuniteit tegen ziekteverwekkers kan verbeteren. Onlangs hebben we ons gericht op de ontwikkeling van regulatie- en geheugen-T...

Video: Auteur in de schijnwerpers: geoptimaliseerd protocol voor het detecteren van antigeenspecifieke T-cellen in muizenlongen met behulp van tetrameren

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Biology

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Biochemistry

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells

Source: Gonzalez-Nieto, L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54881">Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining.</a> J. Vis. Exp. (2016)
This video demonstrates a method for characterizing CD8+ memory T cells specific to simian immunodeficiency virus (SIV) using peptide-major histocompatibility complex class I (pMHC-I) tetramers. A pMHC-I tetramer is a complex of four MHC-I molecules bound to SIV-specific peptides and conjugated to a fluorophore. These tetramers selectively bind to T cell receptors (TCRs) on SIV-specific CD8+ T cells, facilitating the characterization of these cells.

1. PKI Treatment
NOTE: This is optional, but recommended for Mamu-B*017:01 tetramers.

	Resuspend PBMC in R10 medium at a concentration of 1.6 x 107 ...

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

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