**3. Immunological characterization of allergen-specific T cells**

There are several approaches to isolate allergen-specific cells ex vivo for subsequent downstream immunological profiling using technologies, such as RNA or TCR sequencing. These technologies have become of increasing importance in areas, such as biomarker discovery or developing tools to monitor the efficacy of allergen-specific immunotherapy.

#### **3.1. MHC tetramer assay**

reactivity, using peripheral blood mononuclear cells (PBMCs) from allergic patients, often after in vitro expansion with allergen or allergen extract [22, 23]. Peptides that elicit T cell reactivity, as measured by cytokine production, proliferation, or upregulation of T cell activation markers, are reported as T cell epitopes. T cell epitope mapping using overlapping peptides is a very thorough approach, designed to identify any possible T cell-activating region within the allergen. However, mapping peptides for bigger allergens or even multiple allergens can add up to a very high number of peptides to test, also increasing the amount of blood needed for screening and the cost and effort associated. To make large-scale epitope identification more feasible, an approach was developed that involves preselection of peptides based on their ability to bind human MHC class II molecules. MHC molecules have a relatively broad specificity for peptide binding. The three-dimensional structure forms a binding cleft that can bind peptides of varying length, typically ranging from 15 to 25 amino acids [24, 25]. The capacity of a peptide ligand to bind MHC class II molecules can be quantitatively measured directly by assessing its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules [26]. However, such experiments are labor intensive and expensive; therefore, computational tools are continuously being developed to model and predict peptide-MHC binding [27, 28]. Using predicted peptide binding as a preselection criterion to decrease the number of peptides to screen for T cell epitope identification is less thorough than using overlapping peptides and may therefore increase the risk of missing T cell-reactive peptides. However, it has been reported that it is a reliable approach to identify the vast majority of T cell epitopes [28, 29], and it has been successfully used in several allergen systems, including Timothy grass [11], German cockroach [30], house dust mite [31], and others [32], to perform large-scale epitope identification studies. Therefore, the decision between using overlapping and predicted peptides is likely dictated by the size and number of allergens studied as well as the amount of

Another challenging aspect of T cell epitope identification in allergy is the low frequency of allergen-specific T cells. A study that evaluated the ex vivo frequency of T cells specific for Fel d 1, the major cat allergen, reported that the percentage of CD4+ T cells specific for a single Fel d 1 epitope ranged from 0.014 to 0.0003% in allergic individuals [33]. Another study, focused on Mugwort allergy, reported an ex vivo frequency of peptide-specific T cells of 0–0.029% in allergic cohort [34]. In a third study, performed with cells from patients allergic to Timothy grass, the authors reported epitope-specific T cell frequencies of 0.6–0.75% of the total CD4+ T cell subset [35], with a modest increase in frequencies detected during grass pollen season. The rarity of allergen-specific T cells poses a great challenge for epitope identification, as it will require the T cell reactivity assay to reliably detect a few single cells that respond to the peptide among several thousands of CD4+ T cells. In addition, a large amount of blood volume would be required to screen a given number of peptides. To bypass this problem, in vitro expansion cultures are performed, in which lymphocytes from allergic individuals are cultured over a few days or weeks with allergen extract or recombinant allergen protein to which the donor is allergic. The allergen in the culture will activate and stimulate the few antigen-specific T cells

cells available from the clinical cohort.

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**2.2. Allergen-specific T cell frequencies**

The use of MHC tetramer reagents to detect antigen-specific T cells is a well-established technique that allows detection and further downstream analysis of allergen-specific cells on a single cell level. The tetramer molecule is made up of a fluorescently labeled, centric streptavidin molecule bound to biotin-labeled MHC molecules, which are loaded with a peptide known to be a T cell epitope to form the peptide-MHC complex (**Figure 1A**) [38]. The resulting tetramer can then be used as a reagent to bind T cells that are specific for both, the MHC type and peptide used in the tetramer (**Figure 1B**). Cells that are specific and bind the tetramer are now fluorescently labeled and can be detected and isolated using a flow cytometer. There are several applications for tetramer staining all based on the premise that it allows the detection of single antigen-specific cells, even if they occur at low frequency. In vaccinology, tetramers are often used to track frequencies of peptide-specific T cells in the blood before and after vaccination or boost. Similarly, in allergy, tetramers have been used to quantify numbers of specific T cells as a variable of allergen season [35], allergen-specific immunotherapy [39], and disease status [34, 40]. In addition, tetramer staining can be combined with other methodologies to perform more detailed immunological characterization of allergen-specific T cells. Simultaneous assessment of cell proliferation, cytokine production, or activation can provide

**Figure 1.** (A) Schematic representation of the structural complex of a MHC class II tetramer and (B) binding of tetramer molecules to the peptide-specific T cell via the T cell receptor (TCR).

functional information in addition to knowing the peptide specificity and MHC restriction of the cell. Tetramer reagents can even be used for T cell epitope mapping. This approach involves loading empty MHC molecules with pools of mixtures of overlapping peptides from the allergen of interest, each pool typically containing 5–10 peptides. These tetramers are then screened with PBMC that have been cultured with the allergen of interest. Pools that positively detect T cell populations are deconvoluted into single peptides, which are loaded onto MHC molecules individually and then analyzed to identify single epitopes. Tetramers that return positive stainings automatically provide a population of T cells with a known MHC restriction and antigen specificity, which can be sorted by fluorescence-activated cell sorting (FACS) allowing downstream analysis of phenotype and genotype. This approach has successfully been used in allergy to identify T cell epitopes [41].

MHC tetramer assays represent a revolution for the study of antigen-specific T cells, providing an efficient way to directly visualize, quantify, phenotype, and isolate T cells of interest. Yet, this technology is also associated with disadvantages and limitations. The construction of tetramer reagents is not trivial and requires an advanced level of expertise. Production and purification of high-quality MHC molecules are labor intensive, and only a subset of MHC alleles expressed by humans is available as tetramer. Further, the use of tetramers requires existing knowledge about the HLA restriction of the peptide of interest. In allergy, many dominant T cell epitopes are highly promiscuous, meaning they are restricted by multiple alleles, which makes finding their restriction more difficult. Determining the HLA restriction of given peptides can be done experimentally, for example, by inhibition with locus-specific antibodies [32]. This method, however, only identifies the restricting locus. Data from HLA-binding assays can also be a useful tool to narrow down the possible restriction [42]. Another approach designed to determine HLA restriction at the allele level involved the use of single HLA class II-transfected cell lines [43]; however, a large panel of cell lines is required to determine restrictions in multiple donors due to the heterogeneity of HLA types in a given population. As an alternative to the experimental approaches, which are labor intensive and technically challenging, a bioinformatical prediction tool was developed. This tool uses T cell response data in an HLA-typed population to infer HLA restriction by genetic association [44]. Although this tool streamlines the prediction of HLA restriction, it still requires experimental T cell response data and an HLA-typed population large enough to make significant predictions possible.

The use of tetramer reagents requires preexisting knowledge about the HLA restrictions for a given peptide as well as the HLA type of the donor sample. Acquiring this information can be costly and labor intensive, making this approach less feasible for certain studies.

#### **3.2. Cytokine capture assay**

functional information in addition to knowing the peptide specificity and MHC restriction of the cell. Tetramer reagents can even be used for T cell epitope mapping. This approach involves loading empty MHC molecules with pools of mixtures of overlapping peptides from the allergen of interest, each pool typically containing 5–10 peptides. These tetramers are then screened with PBMC that have been cultured with the allergen of interest. Pools that positively detect T cell populations are deconvoluted into single peptides, which are loaded onto MHC molecules individually and then analyzed to identify single epitopes. Tetramers that return positive stainings automatically provide a population of T cells with a known MHC restriction and antigen specificity, which can be sorted by fluorescence-activated cell sorting (FACS) allowing downstream analysis of phenotype and genotype. This approach has suc-

**Figure 1.** (A) Schematic representation of the structural complex of a MHC class II tetramer and (B) binding of tetramer

MHC tetramer assays represent a revolution for the study of antigen-specific T cells, providing an efficient way to directly visualize, quantify, phenotype, and isolate T cells of interest. Yet, this technology is also associated with disadvantages and limitations. The construction of tetramer reagents is not trivial and requires an advanced level of expertise. Production and purification of high-quality MHC molecules are labor intensive, and only a subset of MHC alleles expressed by humans is available as tetramer. Further, the use of tetramers requires existing knowledge about the HLA restriction of the peptide of interest. In allergy, many dominant T cell epitopes are highly promiscuous, meaning they are restricted by multiple alleles, which makes finding their restriction more difficult. Determining the HLA restriction of given peptides can be done experimentally, for example, by inhibition with locus-specific antibodies [32]. This method, however, only identifies the restricting locus. Data from HLA-binding assays can also be a useful tool to narrow down the possible restriction [42]. Another approach designed to determine HLA restriction at the allele level involved the use of single HLA class II-transfected cell lines [43]; however, a large panel of cell lines is required to determine restrictions in multiple donors due to the heterogeneity of HLA types in a given population. As an alternative to the experimental

cessfully been used in allergy to identify T cell epitopes [41].

molecules to the peptide-specific T cell via the T cell receptor (TCR).

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The isolation of antigen-specific cells based on cytokine production used to be complicated by the fact that T cells positive for cytokine production were detected by intracellular cytokine staining, which involved fixation and permeabilization of the cell. Fixed cells are no longer alive and can therefore not be used for downstream applications that require live cells, and even isolation of DNA or RNA from fixed cells is somewhat more complex than from live cells. A new approach that captures cytokines on the cell surface immediately after secretion was developed to allow detection and isolation of viable cells that secrete cytokines in response to antigen stimulation. In this protocol, cells are pre-labeled with a "catch reagent," a divalent complex consisting of a CD45-specific monoclonal antibody conjugated to monoclonal antibody directed against the cytokine of interest. The anti-CD45 antibody will bind to CD45 molecules expressed on the T cell surface and effectively coat the cell (**Figure 2**). Subsequently, cells are stimulated with antigen, and any cytokine produced will be bound to the cytokine-specific antibody conjugated to anti-CD45 immediately after secretion. Detection of cytokine-positive cells is achieved by using a fluorescent-labeled detection antibody with the same cytokine specificity but recognizing a different epitope from the catch reagent antibody (**Figure 2**). If the antigen-specific T cell population is extremely rare, which is often the case in allergy and asthma, an enrichment step can be performed. To further enrich antigen-specific cells before flow cytometric analysis or isolation, microbeads conjugated to monoclonal antibodies specific for the respective fluorophore used in the experiment can be used to label cells, followed by magnetic column enrichment [35]. After cells are labeled and the enrichment step has been performed if desired, viable cells can be analyzed and isolated by flow cytometry, facilitating downstream applications, such as further culture assays or DNA/RNA extraction for sequencing analysis. A potential limitation of this assay is the bias introduced by isolating cells based on production of a single cytokine. Often, cytokine production in response to allergens is heterogeneous, and cells produce different levels of different cytokines, such as IL-4, IL-5, IL-13, and sometimes IFNg. Detection of allergen-specific cells based on production of a single cytokine will likely lead to an underrepresentation of allergen-specific cells, since cells producing a different cytokine will not be detected.

#### **3.3. Cell activation assays**

Another hallmark of antigen-specific T cells is the upregulation of activation markers in response to antigen stimulation. Therefore, these activation markers can be targeted with

**Figure 2.** A schematic representation of the methodology involved for a cytokine capture assay, using IL-5 as representative cytokine.

fluorescent antibodies to detect antigen or allergen-specific cells. The challenge of this approach is to identify activation markers that are specific and highly expressed to allow reliable detection of allergen-specific T cells even at low frequency. One molecule that has become very popular for such an application is CD154, also known as CD40 ligand (CD40L). CD154 is a member of the tumor necrosis factor (TNF) superfamily and found to be primarily expressed on activated T cells, making it very specific. It acts as a co-stimulatory molecule, binding to CD40 on antigen-presenting cells, which can lead to several downstream events depending on the target cell type. Several studies designed to study allergen-specific effector cells in cohorts suffering from allergy, asthma, or who have been treated with AIT have successfully applied this methodology to immunologically characterize and isolate allergen-specific T cells ex vivo [37, 45]. The caveat of using CD154 as a selection marker for activated, allergen-specific T cells is that it is also typically stained intracellularly. In humans, CD154 molecules expressed on the cell surface quickly become unstable, making a large number of CD154 expressing cells undetectable. Therefore, this assay typically involves fixation and permeabilization to allow intracellular staining of CD154, making downstream applications less feasible.

As an alternative to CD154, other activation markers, such as Ox40 and CD25 have also been used to detect and isolate antigen-specific cells after short-term antigen stimulation [46]. The main advantage of this approach is that both Ox40 and CD25 are stably expressed on the cell surface and therefore cells can be detected and isolated in viable form without the need of fixation or permeabilization. However, CD25 is also strongly expressed by regulatory T cells, irrespective of activation; therefore, gating of Ox40 and CD25 double-positive cells has to be performed with great accuracy, and the inclusion of a third marker, such as PDL-1 may be considered to avoid contamination of nonspecific T cells.

#### **3.4. Proliferation assays**

fluorescent antibodies to detect antigen or allergen-specific cells. The challenge of this approach is to identify activation markers that are specific and highly expressed to allow reliable detection of allergen-specific T cells even at low frequency. One molecule that has become very popular for such an application is CD154, also known as CD40 ligand (CD40L). CD154 is a member of the tumor necrosis factor (TNF) superfamily and found to be primarily expressed on activated T cells, making it very specific. It acts as a co-stimulatory molecule, binding to CD40 on antigen-presenting cells, which can lead to several downstream events depending on the target cell type. Several studies designed to study allergen-specific effector cells in cohorts suffering from allergy, asthma, or who have been treated with AIT have successfully applied this methodology to immunologically characterize and isolate allergen-specific T cells ex vivo [37, 45]. The caveat of using CD154 as a selection marker for activated, allergen-specific T cells is that it is also typically stained intracellularly. In humans, CD154 molecules expressed on the cell surface quickly become unstable, making a large number of CD154 expressing cells undetectable. Therefore, this assay typically involves fixation and permeabilization to allow intracellular staining of CD154, making downstream

**Figure 2.** A schematic representation of the methodology involved for a cytokine capture assay, using IL-5 as repre-

As an alternative to CD154, other activation markers, such as Ox40 and CD25 have also been used to detect and isolate antigen-specific cells after short-term antigen stimulation [46]. The main advantage of this approach is that both Ox40 and CD25 are stably expressed on the cell surface and therefore cells can be detected and isolated in viable form without the need of fixation or permeabilization. However, CD25 is also strongly expressed by regulatory T cells, irrespective of activation; therefore, gating of Ox40 and CD25 double-positive cells has to be performed with great accuracy, and the inclusion of a third marker, such as PDL-1 may be

applications less feasible.

sentative cytokine.

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considered to avoid contamination of nonspecific T cells.

The identification of antigen-specific cells based on the proliferative response to antigenic stimulation is perhaps the most classical approach and has been widely used for several applications including T cell epitope mapping, phenotypic characterization, T cell response kinetics, and others. In the past, the classic method to detect cell proliferation in response to allergen stimulation involved the addition of radioactive nucleoside, 3H-thymidine, to the culture, which would be automatically incorporated into new strands of chromosomal DNA during mitotic cell division. Subsequently, proliferation was assessed by measuring the radioactivity in DNA recovered from the cell sample using a scintillation beta-counter. Though this technology is still used in some laboratories, proliferation is now more commonly detected by flow cytometry. One common approach is the staining of cells with a special fluorescent dye, which is then diluted through each cell division. This decrease in the concentration of the dye can be visualized by flow cytometry. Another approach is to stain stimulated cells with antibodies targeting markers associated with proliferation, such as Ki67. The measure of proliferation in response to antigen stimulation is straightforward and inexpensive. The greatest caveat associated with using proliferation as a readout for antigen-specific reactivity is the relatively high rate of false positivity due to bystander activation. A study designed to directly compare the use of tetramer staining reagents versus allergen-induced proliferation for the detection of allergen-specific T cells found that while tetramers had a relatively low rate of sensitivity, cells identified based on proliferation contained extremely high fractions of bystander cells [34], making this approach more suitable if an enriched population is sufficient for the study rather than a desire for a pure antigen-specific population.
