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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+peripheral+fates+of+tissue-restricted+self+antigen-specific+conventional+and+regulatory+CD4(+)+T+cells.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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++ or Mg++</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS without Ca++ or Mg++</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&#8217;s Ham&#8217;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&#39;s Balanced Salt Solution with Ca++ or Mg++</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

819 Views.  Massachusetts General Hospital. 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.

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

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.

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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

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 ...

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 ...