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 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.
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, 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 "peptide:MHC tetramers"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.
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. Please click here to view a larger version of this figure.
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
2. Generating single-cell suspension from lung tissue via automated tissue dissociator
3. Alternative protocol: Generating single-cell suspension from lung tissue via manual dissociation
4. Enriching the lung sample for T cells via magnetic bead enrichment
5. Staining the antigen-specific T cells with peptide:MHC tetramers
6. Analyzing the tetramer-labeled T cells by flow cytometry
Fluorochrome | Antibody |
BUV395 | CD90.2 |
Pacific Blue | CD45 (added earlier by i.v. injection) |
Pacific Orange | CD8 |
Brilliant Violet 785 | CD4 |
FITC | CD3 |
PerCP-Cy5.5 | Dump (B220, CD11b, CD11c, F4/80) |
PE | pMHC tetramer or phenotypic marker |
PE-Cy7 | Phenotypic marker (e.g. CD44, PD-1, CD69, etc.) |
APC | pMHC tetramer or phenotypic marker |
AlexaFluor 700 | Phenotypic marker (e.g. CD44, PD-1, CD69, etc.) |
APC-Cy7 | Live/dead stain |
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
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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'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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 (>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.
The authors have nothing to disclose.
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.).
100 mm cell strainer | Fisher Scientific | 22-363-549 | |
10x PBS without Ca++ or Mg++ | Corning | 46-013-CM | |
1x PBS without Ca++ or Mg++ | Corning | 21-031-CV | |
AccuCheck Counting Beads | Invitrogen | PCB100 | |
Aminoguanidine Hemisulfate Salt | Sigma-Aldrich | A7009 | |
CD90.2 microbeads, mouse | Miltenyi | 130-121-278 | |
Cell separation magnet (MidiMACS Separator) | Miltenyi | 130-042-302 | Holds single LS column |
Cell separation magnet (QuadroMACS Separator) | Miltenyi | 130-090-976 | Holds 4 LS columns |
Dasatinib | Sigma-Aldrich | CDS023389 | |
DNase I | Roche | 10104159001 | |
Eagle’s Ham’s Amino Acids medium | Sigma-Aldrich | C5572 | |
gentleMACS | Miltenyi | 130-093-235 | Automated tissue dissociator |
gentleMACS C Tubes | Miltenyi | 130-093-237 | Automated tissue dissociator tubes |
Hank's Balanced Salt Solution with Ca++ or Mg++ | Corning | 21-020-CM | |
HEPES | Gibco | 15630080 | |
Ketamine | Vedco | NDC 50989-996-06 | |
Liberase TM | Roche | 5401119001 | |
Pacific Blue anti-mouse CD45 antibody (Clone: 30-F11) | Biolegend | 103126 | |
Paramagnetic cell separation columns (LS Columns) | Miltenyi | 130-042-401 | Comes with plunger |
Purified anti mouse CD16/32 antibody (Clone: 93) | Biolegend | 101302 | |
RPMI 1640 medium without L-glutamine | Corning | 15-040-CM | |
Sodium Chloride 0.9% (Normal Saline) | Cytiva | Z1376 | |
Xylazine | Pivetal | NDC 466066-750-02 |