This work was supported by Public Health Service grants from the National Institutes of Health (T32 DA007097, R01AI096966, andUM1AI26617).

1. Day 1: Fresh Tissue Sectioning and Primary Incubation
<ol>
	<li>Use a scalpel to cut fresh tissue into small (approximately 0.5 cm wide by 0.5 cm tall) pieces. Separately glue each tissue to a plunger and embed them with 3 - 5 mL of 4% low-melt agarose in PBS. Label the plunger with the tissue information using a sticker. Put it in a chilled holder in an ice bucket to solidify.</li>
	<li>Turn on the microtome and set the thickness of the sections to 200 &#181;m. Install a razor blade on the microtome and insert the plunger mounted with tissue in the microtome bath.</li>
	<li>Prepare phosphate-buffered saline with heparin (PBS-H) by adding 100 &#181;g/mL or 18.7 U/mL heparin to PBS to preserve the RNA and to permit potential ISH applications downstream. Fill the microtome bath, covering the embedded tissue, with 100 - 120 mL of chilled, sterile PBS-H. Add PBS-H ice cubes to the bath to maintain the temperature at 0 - 2 &#176;C. Start the microtome and cut the tissue into 200 &#181;m sections. 
	Note: It is important to keep the tissues chilled on ice to minimize cellular activity within the tissues and because fresh tissue is easier to section when it is chilled. PBS alone can be used if there are no plans for downstream ISH.</li>
	<li>Alternatively, for tissues that do not cut well with a microtome (e.g., gut and lung), use a scalpel or razor blade to cut the tissue into thin strips as close as possible to 200 &#181;m.</li>
	<li>Label the lid of 24-well tissue culture plates with the experimental sample information and place the tissue chambers in the corresponding wells. Use a paintbrush to transfer the sections to a tissue chamber set in the well of a 24-well tissue culture plate containing 1 mL of chilled PBS-H. 
	NOTE: Reusable tissue chambers should be made prior to initiating staining. Tissue chambers can be made using a 14 mL polypropylene round-bottom snap-cap tube and wire mesh. Use a sharp razor blade to cut the bottom of a 14 mL polypropylene round-bottom snap-cap tube off. Cut the wire mesh in a circle to fit the hole at the bottom of the tube. Heat the wire mesh circle using a Bunsen burner until it is red-hot. Set the wire mesh circle down very quickly and push the tube onto the mesh. Check that the wire mesh is securely attached to the bottom of the tube and then carefully cut off the top of the tube at the 3 mL mark using a sharp razor blade. Put up to 3 tissue sections into each tissue chamber, or up to 1 cm2 of tissue per well. Keep at least one empty well between wells with different antibody combinations to avoid cross-contamination.</li>
	<li>Proceed to the primary tetramer and antibody staining immediately after finishing the transfer of all cut sections into the tissues chambers. Keep the sections submerged and chilled in 1 mL of PBS-H at all times.</li>
	<li>Incubate the tissue sections overnight with 0.5 &#181;g/mL FITC-conjugated, peptide-loaded MHC-I tetramers diluted in PBS-H with 2% normal goat serum (NGS). Include mouse or other non-rabbit antibodies directed at extracellular epitopes of interest in this incubation (e.g., rat anti-CD8 antibodies diluted 1:500 in PBS-H with 2% NGS). Place 1 mL of diluted antibodies in each well. 
	NOTE: Care should be taken when selecting CD8 antibodies, as some can enhance and some can inhibit MHC tetramers binding to T-cell receptors4,21. The rat anti-human CD8 antibody described here is unstable and sometimes results in somewhat weak staining. It is used here for triple labeling because it is the only non-rabbit and non-mouse CD8 antibody tested that stained rhesus macaque CD8 T cells.</li>
	<li>Use 1 mL of solution per well for the primary antibody and all subsequent incubations, and perform this and all subsequent incubations at 4 &#176;C, with the plates on a rocking platform. 
	NOTE: Tissues should float freely in the chamber.</li>
</ol>
2. Day 2: Fixation and Secondary Incubation
<ol>
	<li>After the primary incubation, wash the sections twice with 1 mL of chilled PBS-H for 20 min each wash. Do this by transferring the tissue chambers to a different 24-well tissue culture plate containing 1 mL of chilled PBS-H in the corresponding wells. 
	NOTE: Be careful not to drip contents from one experimental sample into another when moving between tissue chambers. For all subsequent incubations and washes, similarly transfer the tissue chamber to a clean plate containing the appropriate solution. Be sure to monitor the sections in the tissue chambers during the procedure to make sure that the sections do not get stuck to the sides of the tissue chambers. If they do, push them back into the solution.</li>
	<li>Fix the sections with 1 mL of fresh PBS-buffered 4% paraformaldehyde for 2 h at room temperature (do not over-fix). Wash with cold PBS-H twice for 5 min. 
	Caution: Paraformaldehyde is toxic; wear appropriate personal protective equipment. 
	NOTE: If antigen retrieval and permeabilization is needed to detect intracellular epitopes, antigens can be retrieved by boiling the sections in 0.01 mol urea after the paraformaldehyde fixation.</li>
	<li>Transfer the sections into 24-well culture plates containing 0.01 mol urea and place these plates in a microwave oven. Boil the sections three times for about 10 s each, for a total of 30 s. 
	NOTE: Be very careful, as boiling the solution can force the sections out of the wells. If this happens, use a paintbrush to push the sections from the sides of the tissue chamber or from the lid of the plate back into the bottom of the appropriate tissue chamber.</li>
	<li>Prior to the secondary antibody incubation, permeabilize and block the tissue sections by incubating them in blocking solution containing PBS-H, 0.3% detergent (PBS-H-T), and 2% NGS on a rocker for 1 h at 4 &#176;C. Perform subsequent antibody incubations with PBS-H-T/2% NGS.</li>
	<li>For the secondary incubation, transfer the sections in the tissue chambers to wells containing rabbit anti-FITC antibody diluted 1:10,000 in PBS-H-T/2% NGS. Incubate overnight.</li>
	<li>Perform counterstaining using mouse anti-CD20 antibody diluted 1:200 in PBS-H-T/2% NGS. For this option, retrieve the epitopes if needed, permeate the cells, and block prior to this incubation, as described above.</li>
</ol>
3. Day 3: Tertiary Incubation
<ol>
	<li>After the second incubation, wash the sections three times in PBS-H at 4 &#176;C for at least 20 min.</li>
	<li>Perform a final incubation with the appropriate fluorescently labeled antibodies (e.g., goat anti-rabbit conjugated greenish yellow, goat anti-rat conjugated far red dye, and goat anti-mouse conjugated green dye antibodies diluted 1:5,000, 1:5,000, and 1:2,000, respectively, in PBS-H-T/2% NGS). Incubate overnight. 
	NOTE: At this point, the incubation can be extended for up to three days if needed. Keep the sections protected from light by wrapping the plates in tin foil during this incubation step and thereafter, as light quenches fluorophores.</li>
</ol>
4. Day 4: Mounting the Sections
<ol>
	<li>Wash the sections three times in PBS-H for at least 20 min. 
	NOTE: If planning for downstream ISH19, fix the sections in 4% paraformaldehyde for 1 h to secure the tetramers and antibodies in place and then wash the sections twice with PBS-H for 5 min each. 
	Caution: Paraformaldehyde is toxic; wear appropriate personal protective equipment.</li>
	<li>Use a paintbrush to transfer the sections to a microscope slide. Be careful not to poke the tissue too much. Coat each section with glycerol/gelatin containing 4 mg/mL n-propyl gallate or another mounting medium containing a fluorophore preservative. Cover with a coverslip.</li>
	<li>Store the slides in a light-protected slide container at -20 &#176;C. Rinse the tissue culture plates and remove the labels on the lids using alcohol. 
	NOTE: The plates can be reused.</li>
</ol>
5. Acquisition of Confocal Microscope Images
<ol>
	<li>Capture high-resolution images with a confocal microscope, using the appropriate lasers and filters for each fluorophore (Figure 1A and B). 
	NOTE: In this example, a confocal microscope (see the Table of Materials) was used. Images were collected using the 561 nm laser at 20% power for the greenish yellow-labeled antigen-specific T cells, the 488-nm laser at 10% power for green-labeled CD20-expressing B cells, and the 640-nm laser at 15% power for far red-labelled CD8 T cells. The 20X objective and a numerical aperture of 0.8 were used.</li>
	<li>Sequentially collect z-series at 3 &#181;m (or other) intervals in the three channels in multiple 800 x 800 pixel fields. Construct a montage of the collected fields (Figure 1C-E). Name each montage image based on the information of the corresponding slide and save for analysis.</li>
	<li>Perform quantitative image analysis using the respective confocal microscope analysis and quantification software or using ImageJ.</li>
</ol>
6. Quantitative Image Analysis
NOTE: Quantitative image analysis can be accomplished using confocal microscope analysis and quantification software or by using ImageJ software. Here, ImageJ was used as an example.
<ol>
	<li>Open a confocal montage by dragging it to the ImageJ window (Figure 2A). 
	NOTE: ImageJ can directly open montages collected by many different confocal microscopes. If the montage cannot be directly opened by ImageJ, export the selected z-scan as a TIFF file to open it.</li>
	<li>Duplicate the selected z-scan for analysis (&#34;Image&#34;-&#62; &#34;Duplicate&#34;) (Figure 2B).</li>
	<li>Split the different channels (&#34;Image&#34;-&#62; &#34;Color&#34;-&#62; &#34;Split Channels&#34;) (Figure 2C).</li>
	<li>Draw the ROI for quantitative analysis in the corresponding channel to be objective and add it to the ROI manager by pressing &#34;T&#34; on the keyboard. Measure the area. 
	NOTE: The ROI manager of ImageJ shows the area in &#181;m2 (Figure 2D).</li>
	<li>Adjust the fluorescence brightness and contrast of the channel to be analyzed (&#34;Image&#34; -&#62; &#34;Adjust&#34; -&#62; &#34;Brightness/Contrast&#34;) (Figure 2E).</li>
	<li>Flatten the ROI on the image (&#34;ROI manager&#34; -&#62; &#34;Flatten&#34;) (Figure 2F).</li>
	<li>Quantify the positive cells in the image using the &#34;Multi-point&#34; tool (Figure 2G).</li>
</ol>

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</ol>

The authors have nothing to disclose.

IST combined with IHC provides an essential tool for detecting, characterizing, and quantifying antigen-specific CD8 T cells in native environments with the context of other cells and tissue structures. Here, we described detailed procedures for IST combined with IHC, followed by quantitative image analysis, to determine the location, abundance, and phenotype of antigen-specific CD8 T cells in lymph nodes from rhesus macaques. Similar staining can be applied to human, mouse, or other species tissues for which MHC-I tetra...

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 peptide/MHC class I tetramer staining of antigen-specific CD8 T cells1 and the more recent development of MHC class II tetramer staining of CD4 T cells2 by flow cytometry 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 does not allow for the detection of 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 tissues3.
We and others have developed methods using peptide-loaded MHC class I and class II tetramer or multimer reagents to stain antigen-specific CD8 and CD4 T cells in tissues4,5,6,7,8,9,10,11,12,13. These IST methods allow for the determination of the location, abundance, and phenotype of antigen-specific CD8 and CD4 T cells in tissues and provide a means to detect of these cells relative to other cells and structures in the tissues. Our group has extensively used MHC-I tetramer staining to study human immunodeficiency virus (HIV)- and simian immunodeficiency virus (SIV)-specific CD8 T cells in lymphoid, genital, and rectal tissues to gain an understanding of HIV and SIV immunopathogenesis and to identify correlates of successful vaccination strategies14,15,16,17. In addition, we also developed a technique that combines IST with in situ hybridization (ISH) to localize and quantify virus-specific CD8 T cells and virus-infected cells in tissues and to determine the in vivo effector-to-target levels18,19.
Here, we describe a protocol using peptide-loaded MHC-I tetramers to stain antigen-specific CD8 T cells in fresh tissue sections, to counterstain tissues using IHC, and to quantify cells with specific phenotypes in specific tissue compartments. These procedures are the same as were used in our recent publication by Li et al., in which we determined the location, abundance, and phenotype of SIV-specific T cells in lymphoid tissue during chronic SIV infection in macaques20.
For this procedure, fresh tissues are sectioned and incubated overnight with peptide-loaded MHC-I tetramers conjugated to fluorescein thiocyanate molecules (FITC). They are then fixed in paraformaldehyde. After fixing the tissue, the signal from the MHC tetramers is amplified using rabbit anti-FITC antibodies and incubated with fluorescently tagged anti-rabbit IgG antibodies, which further amplify the signal from the bound tetramers. IHC is used in conjunction with IST to characterize antigen-specific T cells and surrounding cells. Antibodies that recognize epitopes on the surface of cells or in the extracellular space are included in the primary incubation with the tetramers. Antibodies that recognize intracellular epitopes require permeation of the cell wall prior to staining. The stained tissue sections are imaged using a confocal microscope and analyzed using confocal software. Labeled cells are quantified using confocal microscopy software or ImageJ. The described protocol can be used to stain essentially any antigen-specific CD8 T cell in any tissue for which MHC-I tetramers are available.

<table><tbody><tr><td>MHC-I monomer</td><td>NIH tetramer core facility</td><td></td><td>Materials for MHC-tetramer preparation</td></tr><tr><td>ExtrAvidin-FITC</td><td>Sigma-Aldrich</td><td>E2716</td><td>Materials for MHC-tetramer preparation</td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Low melt agarose</td><td>Promega</td><td>V3121</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>SLBL6391V</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-6878</td><td></td></tr><tr><td>Urea</td><td>J.T.Baker</td><td>4204-05</td><td></td></tr><tr><td>Glycerol gelatin</td><td>Sigma-Aldrich</td><td>SLBH2672V</td><td></td></tr><tr><td>n-propyl gallate</td><td>Sigma-Aldrich</td><td>P3130</td><td></td></tr><tr><td>rat-a-h-CD8 (1:500)</td><td>Acris</td><td>0714</td><td>Antibody unstable, use single use frozen aliquot</td></tr><tr><td>m-a-h-CD20 (1:500)</td><td>NOVOCASTRA</td><td>6026819</td><td></td></tr><tr><td>m-a-h-Ki67 (1:500)</td><td>Vector</td><td>6022201</td><td></td></tr><tr><td>goat-a-m-A488 (1:2,000)</td><td>Jackson Immunoresearch</td><td>124083</td><td></td></tr><tr><td>goat-a-rb-Cy3 (1:5,000)</td><td>Jackson Immunoresearch</td><td>106232</td><td></td></tr><tr><td>goat-a-rat-Cy5 (1:5,000)</td><td>Jackson Immunoresearch</td><td>118088</td><td></td></tr><tr><td>goat-a-h-IgM-Dylight649 (1:5,000)</td><td>Jackson Immunoresearch</td><td>86579</td><td></td></tr><tr><td>Compresstome: VF-300 Microtome</td><td>Precisionary Instruments, LLC</td><td>1079</td><td></td></tr><tr><td>Quick Set Instant Adhesive</td><td>Loctite</td><td>46551</td><td></td></tr><tr><td>24-well flat bottomed tissue culture plates</td><td>Falcon</td><td>353226</td><td></td></tr><tr><td>Microscope slide</td><td>Globe scienfitic Inc.</td><td>#1321</td><td></td></tr><tr><td>Razor&amp;nbsp; blade</td><td>Ted Pella, Inc</td><td>121-6</td><td></td></tr><tr><td>Feather Disposable Scalpel</td><td>FEATHER SAFETY RAZOR CO. LTD.</td><td>No. 21</td><td></td></tr><tr><td>Round paintbrush #2</td><td>PRINCETON ART &amp;amp; BRUSH CO.</td><td>4350R</td><td>Can trim as needed with razor</td></tr><tr><td>Confocal Microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>FV10-ASW_Viewer4.0</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

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.

Figure 1 shows how to collect confocal images using a confocal microscope. Figure 2 demonstrates quantitative image analysis using ImageJ. Figures 3 and 4 show representative images of lymph node tissues from an SIV infected rhesus macaque stained with MHC tetramers, CD8 antibodies, and CD20 antibodies, and serve to demonstrate the specificity of the MHC-tetramer staining. Fi...

Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

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.

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 ...

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Research

JoVE Journal

Immunology and Infection

12.2K Views.  University of Minnesota. 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. 

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

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment.
The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

Cancer Research

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

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

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 ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

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 ...

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

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.

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.

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

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

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