3.6 Component-resolved differential diagnosis approach with a single flow cytometry assay
To enhance the component-resolved diagnostic performance of our CytoBas technique for RGP allergy, multiplex flow cytometry was conducted with [Lol p 1]4-APC and [Lol p 5]4-BV711 tetramers in a single tube. Basophils in thawed PBMC from an allergic patient were successfully discriminated from those of a control by a higher MFI for one or both allergen tetramers (Figure 5A-C ). Multiplex flow cytometry on thawed PBMC with both RGP allergen tetramers in a single tube exhibited clear resolution between a series of RGP-allergic patients and controls (Figure 5D ).
To test for discrimination between multiple allergic sensitizations, Api m 1, Lol p 1 and Lol p 5 tetramers, each labelled with a different fluorochrome, were included simultaneously in a single multiplex flow cytometric test. Since the MFI of the allergen tetramers on pDCs was shown to be similar to the MFI of streptavidin control conjugates on basophils, pDCs provided a suitable background control in blood samples for multiplex flow cytometry (Suppl. Fig 5 ). Basophils from thawed PBMC of RGP-allergic patients demonstrated selective binding to only [Lol p 1]4 or [Lol p 5]4tetramers and not [Api m 1]4, whilst basophils from a BV-allergic patient only bound [Api m 1]4(Figure 6A ). The MFI of staining on basophils for each of the three allergens correlated strongly with serum allergen-specific IgE (each p<0.0001; Figure 6B-D ). This is in contrast to the frequency of activated (CD63+) basophils stimulated with Api m 1 and Lol p 1 tetramers, which showed a poorer correlation with Api m 1-specific IgE (r2 = 0.03561, p = 0.2436) or Lol p 1-specific IgE (r2 = 0.1313, p = 0.0114), respectively. The CytoBas technique therefore reflects allergen-specific IgE titers in serum with greater accuracy than basophil activation. Taken together, these data demonstrate that a single flow cytometric assay with multiple allergen tetramers can provide a rapid component-resolved and differential diagnostic test for allergy.
DISCUSSION
We describe here a novel methodology for a component-resolved diagnostic test for allergy using multiplex flow cytometry involving direct staining of basophils with recombinant allergen tetramers – CytoBas. This flow cytometric approach enabled rapid detection with high sensitivity and specificity of BV allergy with Api m 1 and of RGP allergy with Lol p 1. Combined staining of Api m 1 and Lol p 1 reliably enabled differential detection of allergen sensitization, and inclusion of both Lol p 1 and Lol p 5 facilitated quantification of the relative sensitization to distinct components of RGP in a single-tube test. The combination of multiple allergens from a single species provided complete discrimination between RGP-allergic patients and controls, highlighting the utility of multiplex CytoBas in diagnosing allergies that exhibit sensitization to multiple allergen components.
The allergens in our study were recombinantly-produced proteins. This approach facilitates the introduction of mutations that disrupted enzymatic function and the addition of protein tags for biotinylation and purification. Api m 1, Lol p 1 and Lol p 5 contain glycan structures.39,40 Because the glycosylation patterns of proteins in invertebrates and plants are more similar to each other and differ strongly from mammals,41-43 we here produced their recombinant forms in the Sf21 insect cell line. Similar to a previous report utilizing Sf9 cells,44 we obtained two main glycosylated products of the recombinant Api m 1, fitting with the native paucimannosidic type of glycosylation.43 Both recombinant RGP allergens had greater sizes than predicted for non-glycosylated variants, and showed high sensitivity and specificity for detection of RGP allergy. The high sensitivity and specificity of our recombinant allergen components for confirmation of BV and RGP allergies indicates that our approach preserves the major epitopes for IgE binding. The fact that invertebrate- and plant-derived proteins can be generated as such in an insect cell line, as confirmed here, demonstrates the potential to apply our CytoBas assay for detection of sensitization to a huge diversity of food-, aero- and stinging insect-derived allergens.
Our CytoBas study exploits the concept that basophils present high levels of IgE on their surface through FcεRI binding, and that this IgE is polyclonal and reflective of the specificities produced in an individual. This is the same concept that is utilized for BAT, even though there is typically a fraction of cells that does not degranulate and express CD63 after allergen stimulation. Our direct staining approach showed that in the case of allergen-sensitization, all basophils become positive with the population showing a Gaussian distribution of staining intensity. The median staining intensities correlated strongly with allergen-specific serum IgE levels, and so could be used in a similar manner for quantitating the response. Furthermore, the polyclonal nature of IgE on basophils enabled detection of multiple specificities on the total population in a single test.
RGP-allergic patients with Lol p 1 and Lol p 5 co-sensitization showed double-positive basophil populations. This is in contrast to a recent study where distinct basophil subsets were positive for either recombinant Api m 1 or recombinant Api m 2 conjugated to fluorescent quantum dots (Qdots).45 The reason for this difference is unclear, as it is unlikely that within an individual, distinct basophil populations will present unique allergen specificities. Therefore, it will be important for applications of multicolor flow cytometry to incorporate appropriate controls and establish standardized instrument settings for reproducible and robust measurements.37,38
Another novel observation from this study is that degranulated basophils (CD63+) bind allergen tetramers with similar intensity as basophils that have not degranulated (CD63) in the same assay. Typically, the proportion of basophils that degranulate in a BAT forms a bell-shaped curve with increasing concentrations of allergen. The maximum frequency of CD63+ cells will differ between individuals, and the dose-response curve is thought to be affected by the affinity of IgE to the antigen, the density of the epitope-specific IgE on the cell surface, and the functional capacity of the basophil itself.46-48 Our observation argues against differences in basophils from the same individual with regards to IgE affinity or density of epitope-specific IgE. Rather, intrinsic characteristics of the basophil, e.g. signaling threshold, degranulation capacity and maturity, could underlie the differences between those cells that do degranulate and the fraction of cells that does not.
Our allergens were tetramerized using streptavidin-fluorophore conjugates. A previous study has reported that activated basophils may have the capacity to bind streptavidin via positively-charged molecules that are expressed on the basophil surface upon degranulation.49 This could potentially lead to non-specific binding of allergen tetramers to degranulated basophils. In our experiments, we did not observe such an effect and the fluorescence intensities of our allergen tetramers were similar between basophils that had degranulated and those that had not. Still, it is recommended for multiparameter analysis of allergen binding to perform incubations for staining at room temperature or even at 4ºC to minimize the potential for activation and degranulation of basophils that might mediate non-specific binding of streptavidin.
Using a multiparameter staining approach with three allergen components, we demonstrated that CytoBas enabled detection of sensitization to multiple components of the same allergen (RGP; Lol p 1 and Lol p 5) as well as differential sensitization to distinct allergens (BV; Api m 1 vs RGP; Lol p 1 and/or Lol p 5). This differential and component-resolved analysis through staining of basophils has several advantages over BAT: 1) The CytoBas assay only requires direct staining with allergen tetramers and mAbs without the need for in vitro stimulation, making it less dependent on maintaining basophil function ex vivo , less labor intensive and easier to standardize; 2) CytoBas has the potential for a single staining with multiplex detection of distinct allergen components, rather than the need for multiple (7-10) independent conditions for titration of each allergen in the BAT; 3) For BV and RGP allergy, two distinct allergic diseases, CytoBas with Api m 1 and Lol p 1 distinguished between allergic patients and controls with greater sensitivity and specificity than the BAT; 4) Basophil staining with recombinant allergen tetramers is possible on fresh whole blood, fresh PBMC and cryopreserved PBMC enabling more flexibility for transport or batch analysis of clinical samples than is currently possible with the BAT, which requires processing of fresh blood within 24 hrs.50 A current limitation is the availability of recombinant allergens with the correct modifications for tetramerization with fluorescently-labeled streptavidin. However, with the potential for use of insect cells to generate plant and invertebrate proteins, and the vast experience with recombinant proteins in serology-based component resolved diagnostics, this will only be a temporary hurdle.
CytoBas with Lol p 1 tetramers exhibited greater sensitivity and specificity than previously reported for clinical history, SPT and allergen-specific IgE in allergic rhinitis patients sensitized to pollen areoallergens.51 With the potential for multiplex analysis in a single assay, CytoBas has the potential to compete with serology-based IgE tests. Theoretically, a combination of 2-4 major allergen components could be applied to a) detect allergen reactivity in a highly sensitive manner; and b) detect specific sensitization and risk of systemic response, without the need for repeat tests that are currently recommended for e.g. peanut allergy.17 In practice, the composition of a CytoBas assay and its performance will need to be tested and compared to the standard diagnostic workflow for each diagnostic process. The availability of high-end clinical cytometers that allow 10 or more components to be multiplexed in one assay provides the potential for CytoBas to provide a rapid component-resolved diagnostic test for allergy whilst avoiding allergen challenge.