Peanut-specific T cell responses in patients with different clinical reactivity

Abstract

Whole extract or allergen-specific IgE testing has become increasingly popular in the diagnosis of peanut allergy. However, much less is known about T cell responses in peanut allergy and how it relates to different clinical phenotypes. CD4+ T cells play a major role in the pathophysiology of peanut allergy as well as tolerance induction during oral desensitization regimens. We set out to characterize and phenotype the T cell responses and their targets in peanut sensitized patients. Using PBMC from peanut-allergic and non-allergic patients, we mapped T cell epitopes for three major peanut allergens, Ara h 1, 2 and 3 (27 from Ara h 1, 4 from Ara h 2 and 43 from Ara h 3) associated with release of IFNγ (representative Th1 cytokine) and IL5 (representative Th2 cytokine). A pool containing 19 immunodominant peptides, selected to account for 60% of the total Ara h 1-3-specific T cell response in allergics, but only 20% in non-allergics, was shown to discriminate T cell responses in peanut-sensitized, symptomatic vs non-symptomatic individuals more effectively than peanut extract. This pool elicited positive T cell responses above a defined threshold in 12/15 sensitized, symptomatic patients, whereas in the sensitized but non-symptomatic cohort only, 4/14 reacted. The reactivity against this peptide pool in symptomatic patients was dominated by IL-10, IL-17 and to a lesser extend IL-5. For four distinct epitopes, HLA class II restrictions were determined, enabling production of tetrameric reagents. Tetramer staining in four donors (2 symptomatic, 2 non-symptomatic) revealed a trend for increased numbers of peanut epitope-specific T cells in symptomatic patients compared to non-symptomatic patients, which was associated with elevated CRTh2 expression whereas cells from non-symptomatic patients exhibited higher levels of Integrin β7 expression. Our results demonstrate differences in T cell response magnitude, epitope specificity and phenotype between symptomatic and non-symptomatic peanut-sensitized patients. In addition to IgE reactivity, analysis of peanut-specific T cells may be useful to improve our understanding of different clinical manifestations in peanut allergy.

Fig 1. T cell reactivity against peanut allergen-derived peptides.

A) Magnitude (SFC- spot forming cells per 1 PBMC million (i.e. SFC /10⁶)) and frequency (% responders) of IL-5 (open circles) and IFNγ (closed circles) production in response to individual peptides derived from Ara h 1, 2 and 3 were measured by ELISPOT in all tested donors (n = 21). Each dot represents a single peptide that elicited T cell reactivity in one or more donors. B) Magnitude of IL-5 and IFNγ production in allergics (n = 11; open circles) and non-allergic (n = 10; closed circles) patients. The response polarization, expressed as IL-5/IFNγ ratio, is shown for C) all peptides that tested positive in any given donor and D) a set of 19 selected peptides that account for 30% of the total response (60% in allergics, 20% in non-allergics, respectively). Each data point represents a single donor/peptide combination. Statistical comparison by Mann-Whitney test, two-tailed. **- p<0.01, ***-p<0.001, ****-p<0.0001.

Fig 2. Peanut extract and epitope responses and cytokine polarization in peanut symptomatic and non-symptomatic patients.

Magnitude and polarization of cytokine production (sum of IL-5, IL-10, IL-17 and IFNγ) in response to A-B) peanut extract and C-D) P19 pool is shown as SFC (spot forming cells) per 10⁶ PBMC. On panel C, dashed line indicates positive response threshold of 70 SFC. Peanut sensitization status is indicated by IgE+. Sym = symptomatic (n = 15), Non-Sym = non-symtomatic (n = 14). Statistical comparison by Mann-Whitney test, one-tailed. *- p<0.05.

Fig 3. Tetramer staining and phenotypic surface marker expression of T cells in peanut-sensitized donors after in vitro culture.

A) a representative plot showing staining with tetramer and HLA-mismatch control. B) Quantification of tetramer-positive cells in peanut-sensitized, symptomatic (Sym) (n = 2) or non-symptomatic (Non-Sym) (n = 2) donors. In the non-symptomatic cohort, donors were tested with multiple tetramers, therefore a total of 4 data points are shown in this cohort. Median with interquartile range is shown. C) Integrin β7 expression and D) CRTh2 expression in tetramer positive cells from peanut-sensitized, symptomatic and non-symptomatic patients. Left panels show representative FACS plots. Right panels show graphs quantifying Integrin β7 and CRTh2 expression in tetramer+ cells from all samples tested. No statistical analysis was performed due to low sample size.