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. 2016 Aug;138(2):571-578.e7.
doi: 10.1016/j.jaci.2015.11.034. Epub 2016 Feb 13.

T-cell epitope conservation across allergen species is a major determinant of immunogenicity

Luise Westernberg  1 , Véronique Schulten  1 , Jason A Greenbaum  1 , Sara Natali  2 , Victoria Tripple  1 , Denise M McKinney  1 , April Frazier  1 , Heidi Hofer  3 , Michael Wallner  3 , Federica Sallusto  4 , Alessandro Sette  1 , Bjoern Peters  5
Affiliations

T-cell epitope conservation across allergen species is a major determinant of immunogenicity

Luise Westernberg et al. J Allergy Clin Immunol. 2016 Aug.

Abstract

Background: Patients with pollen allergies are frequently polysensitized. Pollens contain epitopes that are conserved across multiple species.

Objective: We sought to demonstrate that cross-reactive T cells that recognize conserved epitopes show higher levels of expansion than T cells recognizing monospecific epitopes because of more frequent stimulation.

Method: RNA was sequenced from 9 pollens, and the reads were assembled de novo into more than 50,000 transcripts. T-cell epitopes from timothy grass (Phleum pratense) were examined for conservation in these transcripts, and this was correlated to their ability to induce T-cell responses. T cells were expanded in vitro with P pratense-derived peptides and tested for cross-reactivity to pollen extracts in ELISpot assays.

Results: We found that antigenic proteins are more conserved than nonimmunogenic proteins in P pratense pollen. Additionally, P pratense epitopes that were highly conserved across pollens elicited more T-cell responses in donors with grass allergy than less conserved epitopes. Moreover, conservation of a P pratense peptide at the transcriptomic level correlated with the ability of that peptide to trigger T cells that were cross-reactive with other non-P pratense pollen extracts.

Conclusion: We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within P pratense, as well as their ability to induce cross-reactive T-cell responses. T cells recognizing conserved epitopes might be more prominent because they can be stimulated by a broader range of pollens and thereby drive polysensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of polysensitization and target multiple pollen allergies.

Keywords: RNA sequencing; T cell; cross-reactivity; epitope; pollen allergy; sequence conservation; timothy grass allergy; transcriptome.

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Figures

Figure 1
Figure 1. Conservation of TG proteins across other pollens
(A) 648 peptides from IgE reactive Phl p allergens listed by IUIS were examined for their conservation in other pollen species. Peptides were considered conserved in another pollen if its transcriptome encoded the peptide or a variant with up to 2 amino acid substitutions. The percentage of peptides conserved in all 4 additional grass transcriptomes (‘pan-grass’) is indicated in light grey bars, and the percentage of peptides conserved in all ten pollens (‘pan-pollen’) in dark grey bars. Antigens are sorted based on pan-grass conservation from low to high. The pan-grass (panel B) and pan-pollen (panel C) conservation of IUIS allergens was then compared to proteins identified in Phl p pollen based on a transcriptomic and proteomic analysis. Median and quantile ranges are indicated by boxes and error bars. Phl p pollen proteins that were not recognized by either T-cell or B cell responses were less conserved than both IUIS allergens and Timothy Grass T-cell antigens (TGTAs). Asterisks indicated statistically significant differences (one-tailed Mann-Whitney test, p < 0.05).
Figure 2
Figure 2. Peptide conservation correlates with immunogenicity
Panels of peptides from TG were previously tested for the ability to induce IL-5 responses in PBMCs from allergic patients after in vitro culture with TG extract in two separate cohorts. Peptides from each study were separated into sets based on the number of pollen species they were conserved in (x-axis). For each set, the average frequency of T-cell responses was calculated and normalized to 1.0 for each study(y-axis). Data for peptides derived from IUIS allergens are shown as blue diamonds, while peptides derived from TGTA antigens are shown as red boxes, and averages of the two are shown as black circles. The line depicts a linear correlation for the averaged data, which is highly significant with r2=0.796.
Figure 3
Figure 3. Conservation in transcriptome predicts peptide cross-reactivity
For each peptide, Phl p allergic donors were selected that reacted to the peptide after expanding PBMCs in vitro with Phl p extract. PBMCs were stimulated with individual peptides for 14 days and IL-5 responses were measured by ELISPOT to i) the peptide itself, ii) Phl p extract, iii) the nine non-Phl p extracts for which transcriptomes were available, iv) peptide pools (P20 and P19) that did or did not contain the peptide, as relevant and irrelevant controls. T-cell cultures that did not induce a robust response above 200 SFC to the peptide itself were excluded (30% of cultures). Reponses to extracts and peptide pools are expressed as the relative fraction of the response to the peptide itself, and capped at 100%. Each grey bar represents the average response +/− SEM. Asterisks indicate p-values of statistical significance as indicated on the right according to one-tailed Mann-Whitney tests.
Figure 4
Figure 4. T cell clone cross-reactivity
Donor 1583 was cultured with peptide P5 and then restimulated with that same (relevant) peptide, an irrelevant peptide control and peptides from the 4 grasses (green), 4 trees (blue) and 2 weeds (red). A) Elispot data from the P5 cultured cell line. B)-E) Proliferation of four representative T-cell clones derived from the same patient.
Figure 5
Figure 5. Schematic representation of allergen cross-reactive vs. mono-specific T-cell epitope recognition
Allergen 1 and 2 contain a conserved T-cell epitope. Cross-reactive T-cells that respond to this epitope will therefore be primed more frequently (cross-reactive T-cell in red, top) than T-cells that are specific for an epitope that is unique to a single allergen (allergen mono-specific T-cell in blue, bottom). Consequently, cross-reactive T-cells can provide T-cell help to B cells that are specific to different allergens, and promote antibody production / isotype switching to IgE. Importantly, both B-cells that produce cross-reactive IgE and B-cells that produce mono-specific IgE can receive help from cross-reactive T-cells as long as they process and present a conserved T-cell epitope.
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