staining the antigen-specific t cells with peptide:mhc tetramers for flow cytometry analysis and sorting

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

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/files/ftp_upload/66939/66939fig01large.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

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

Our lab studies the development of CD4 T cells in different contexts of tolerance and immunity. Across our projects, we are primarily interested in how antigen exposure drives the development of phenotypic subsets of T cells that establish immune memory that can enhance tolerance to itself or immunity against pathogens. Recently we have focused on the development of regulatory and memory T-cell subsets that reside in lung tissue.These tissue resident T cells play an important role in reinforcing tolerance to self-antigens during tissue injury, or promoting inflammatory responses to pathogens during reinfection. Our lab specializes in the direct study of antigen-specific T cells, but custom peptide MHC tetramer reagents. This allows us to study these T cells without the use of TCR transgenic models or other experimental manipulations that are known to cause experimental artifacts.Although we can readily identify antigen-specific T cells with tetramers, these T cells are often present at very low frequencies, particularly when they're in a naive or memory state. It is also challenging to isolate these T cells from non-lymphoid tissues where they're often reside following an immune response. While there are several established protocols for isolating T cells from mouse lungs, the protocol we present here has been optimized for the subsequent detection of low frequency antigen-specific T cells by tetramer staining.Within this protocol, we have also included two options for tissue association to better accommodate library resources. Our protocol will help researchers expand the use of tetramers to antigen-specific T cells in lung tissue. This will lead to more powerful in vivo studies of tissue resident memory T cell development and function, which are topics of intense interest in the field.

Staining the Antigen-Specific T Cells with Peptide:MHC Tetramers for Flow Cytometry Analysis and Sorting

After enriching the lung sample for T cells, add one milliliter of 10 millimolar dasatinib to each sample. Vortex the mixture and incubate for five minutes at room temperature. Add PE or APC conjugated peptide MHC tetramer to a final concentration of 10 nanomolar.After vortexing the mixture, incubate it in the dark for one hour at room temperature. While the sample is stained with peptide MHC tetramers, prepare a master mix of antibodies to stain the cell surface markers with 15 minutes remaining in the tetramer staining incubation period, add the surface antibody master mix at a 1 to 100 dilution. Add 50, 000 flow cytometry count beads and cold sorter buffer to reach a final volume of approximately five milliliters, then add cold sorter buffer to a volume of 15 milliliters and centrifuge the tube at 400 x g for five minutes at four degrees Celsius.Carefully aspirate the supernatant, leaving approximately 100 microliters of the supernatant and the cell pellet. Now Resuspend the sample with an additional 200 microliters of sorter buffer and transfer it to a five milliliter FACS tube.

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Research

JoVE Journal

Immunology and Infection

62 Views. After enriching the lung sample for T cells, add one milliliter of 10 millimolar dasatinib to each sample. Vortex the mixture and incubate for five minutes at room temperature. Add PE or APC conjugated peptide MHC tetramer to a final concentration of 10 nanomolar.After vortexing the mixture, incubate it in the dark for one hour at room temperature. While the sample is stained with peptide MHC tetramers, prepare a master mix of antibodies to stain the cell surface markers with 15 minute...

Video: Staining the Antigen-Specific T Cells with Peptide:MHC Tetramers for Flow Cytometry Analysis and Sorting

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.

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 ...

Generating Single-Cell Suspension from Lung Tissue for Identifying Rare Antigen-Specific T-Cell Populations

Inject one to three milligrams of fluorophore-conjugated anti-mouse CD45 antibody diluted in 100 milliliters of normal saline intravenously into each anesthetized mouse. Begin by placing four milliliters of ice-cold HEPES buffer prepared in a specialized tissue dissociator tube on ice for each mouse. After euthanizing the mouse, place the resected lungs in their respective tissue dissociator tube, chilled on ice.Place the tubes onto the automated tissue dissociator and run the first preset program for lung tissue. Then, add 500 microliters of liberase stock solution and 500 microliters of aminoguanidine stock solution to each tube containing the dissociated lung tissue. Incubate it on a nutating mixer for 30 minutes at 37 degrees Celsius.Afterward, place the tubes back on the dissociator and run the second preset program for lung tissue. Now pour the single-cell suspension from the tissue dissociator tube through a 100-micrometer cell strainer into a 50-milliliter conical tube. Rinse the tissue dissociator tube with five milliliters of complete EHAA and pass through the strainer into the 50-milliliter tube.After removing the strainer, raise the volume of the cell suspension to 50 milliliters with complete EHAA. Instead of using specialized tissue dissociator tubes, place the resected lungs in their respective 1.5 milliliter-microfuge tubes chilled on ice. Once all the lungs have been harvested, transfer each lung to a fresh microfuge tube without any cell media.Using scissors, cut the lungs into small fragments within the microfuge tube. Add one milliliter of liberase TM and DNase I digestion cocktail to each tube. Incubate for 30 minutes at 37 degrees Celsius in a water bath.Then, pour the sample over a 100-micrometer cell strainer placed on top of a 50-milliliter conical tube. Pipette out remaining sample onto the strainer. Mash the tissue against the mesh with the rubber end of the plunger from a sterile one-milliliter syringe.Rinse the strainer with cold sorter buffer, collecting a final volume of seven milliliters in the tube. Whether using the automated or manual dissociation method, centrifuge the cell suspension obtained. Carefully aspirate the supernatant, leaving behind approximately 100 microliters of supernatant and the cell pellet.Finally, add approximately 100 microliters of Fc block solution to bring the total volume up to 200 microliters and vortex vigorously to resuspend the pellet.

Enriching the Lung Sample for T Cells via Magnetic Bead Enrichment

To begin, add 50 microliters of anti-mouse CD90.2 microbeads to the isolated lung cells. After vortexing the mixture, incubate it for 10 minutes at four degrees Celsius. Next, place a paramagnetic cell separation column onto a one or four position cell separation magnet.Position an open 15 milliliter conical tube under each column to capture the flow through. Prime the column with three milliliters of cold sorter buffer, allowing the column to drain by gravity into the conical tube below. After the cells have incubated with the microbeads for 10 minutes, add cold sorter buffer to a volume of one milliliter and transfer the mixture through a 100 micrometer cell strainer placed atop the column.Collect the column flow through into a fresh 15 milliliter conical tube placed below the column. Once the cell suspension has completely drained through the column, add an additional three milliliters of cold sorter buffer to the original tube and apply it to the column through the mesh before removing the strainer. When the sample has completely drained into the column and no further flow through is exiting, remove the column from the magnet and place it atop a fresh 15 milliliter conical tube.Add five milliliters of cold sorter buffer to the column. To elute the bound cells, push the column plunger down in one continuous motion, forcing the buffer out of the bottom into a new tube. Centrifuge the eluted samples at 400 x g for five minutes at four degrees Celsius.Carefully aspirate the supernatant, leaving approximately 100 microliters of the supernatant and the cell pellet. On average, the enrichment process recovers more than 99%of all CD90.2 positive T cells from the lungs with greater than 90%purity.