**2. Challenges of T cell epitope mapping**

on skin prick testing [1], which typically involves pricking the skin with a needle or pin containing a small amount of allergen [2]. A second diagnostic test is commonly performed in vitro for allergen-specific immunoglobulin E (IgE), which can accurately evaluate and quantify the presence or absence of IgE specific for the whole allergen extract or single protein

The importance of IgE in mediating allergic disease, especially immediate-type reactions occurring within minutes of exposure to the allergen, is evident. However, the involvement of allergen-specific T cells and their pathological role in mediating late-phase reactions [4, 5] is often underappreciated. Allergenic proteins are defined based on their ability to bind IgE and the frequency of allergic patients harboring specific IgE antibodies to a given allergen [6, 7]. The potential of an allergenic protein to induce T cell reactivity is mostly not taken into account when classifying a protein as an allergen. Over the past decades, however, the contribution of T cells, specifically T helper 2 (Th2) cells, in mediating the pathogenesis of allergy has been extensively studied [8]. Immunological studies have shown that T cells play a key role early on, before allergic disease is even established. Susceptible individuals initially exposed to allergen mount a dominant Th2 response, resulting in the production of type 2 cytokines, such as IL-4 and IL-13. These cytokines along with a direct physical interaction of T and B cells occurring between CD40L expressed on the surface of the activated T cell and CD40 constitutively expressed by B cells provide the signal for B cells to undergo antibody class switching and produce allergen-specific IgE [9, 10], a process referred to as allergic sensitization. Subsequently, IgE molecules now present in high abundance bind with high affinity to Fcε receptors expressed on granulocytes, where they are cross-linked by allergen molecules upon reexposure, leading to mediator release and immediate-type symptoms, such as urticarial, allergic rhinitis, and conjunctivitis. Immediate-type reactivity is followed by late-phase reactions, which typically occur several hours/days after exposure to allergen. During the late-phase reaction, the affected tissue is infiltrated by Th2 cells and other inflammatory cells including eosinophils and neutrophils, which secrete high levels of cytokines, such as IL-4

T cells are not only significant for the onset and maintenance of allergic disease but likely also play a key role for the induction of tolerance, which can be achieved by allergen-specific immunotherapy (AIT) and is the only curative treatment for allergic disease to date. Due to the complexity of human T cell responses against allergens, epitopes have only been thoroughly mapped for the most dominant and prevalent allergens. Recently developed laboratory approaches enable us to perform thorough peptide screens, which achieve the identification and immunological characterization of T cell epitopes in known and novel allergenic targets, irrespective of their IgE reactivity [11, 12]. Mapping of T cell epitopes is of high importance: it greatly facilitates the detection, immunological analysis, and phenotypic characterization of allergen-specific T cells in patients suffering from allergic or asthmatic disease as well as providing a tool to monitor the efficacy of allergen-specific immunotherapy (AIT) treatment. While allergen extracts can also be used to stimulate allergen-specific T cell responses, extracts are not standardized resulting in great variability of allergen content between extract

components [3].

124 Allergen

and IL-5 to promote inflammation [8].

The identification of T cell epitopes from major allergens is an important goal in allergy research. A critical step for inducing a T cell response against an allergen is the recognition of allergen-derived peptides. These peptides are presented to the T cell by antigen-presenting cells (APCs), such as dendritic cells or monocytes, in the context of major histocompatibility complex (MHC) class II molecules, which are constitutively expressed by APCs. MHC class II molecules are encoded by three different loci, designated HLA DR, DQ, and DP. Each of these three loci is extremely polymorphic adding a high degree of complexity, which has to be accounted for in the design of T cell epitope mapping strategies [21].

#### **2.1. Overlapping versus predicted peptide**

To identify T cell epitopes in allergy, the most diligent approach involves testing overlapping peptides that span the entire sequence of the allergen of interest. For this setup, the entire allergen sequence is broken down into short peptides, typically 12–20 amino acids in length, overlapping by 9–12 residues. These peptides are then tested for their ability to induce T cell 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 cells available from the clinical cohort.

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

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 present in the culture, causing them to proliferate. Typically, recombinant human IL-2 is added in limiting dilution in regular intervals after the first few days of culture to increase proliferation of allergen-specific cells, which have upregulated their IL-2 receptor during cell activation. Over time, allergen-specific cells, which were rare in the starting culture, become highly enriched due to antigen-specific stimulation and proliferation. After several days, the cells can be harvested in screened for T cell reactivity in response to restimulation with single peptides. In the presence of allergen or whole allergen extract, allergen peptide-specific T cells will have expanded and are now present in high abundance, making them easily detectable after restimulation with single peptides. T cell reactivity I response to a peptide can be measured by a variety of assays, most commonly using proliferation, cytokine production, or upregulation of activation marker as a readout [22, 36, 37]. This method is extremely useful to expand very rare antigen-specific CD4+ populations. However, one major limitation associated with in vitro expansion culture is that it changes the original phenotype of the cells. Therefore, it cannot be performed if an immunological characterization of the phenotype of the antigen-specific cells is desired. Analyses designed to investigate the genetic expression profile have to be performed on cells isolated directly ex vivo, which is difficult due to their aforementioned rarity in the peripheral blood.
