Lack of allergy to timothy grass pollen is not a passive phenomenon but associated with the allergen-specific modulation of immune reactivity.

BACKGROUND: Timothy grass (TG) pollen is a common seasonal airborne allergen associated with symptoms ranging from mild rhinitis to severe asthma. OBJECTIVE: The aim of this study was to characterize changes in TG-specific T cell responses as a function of seasonality. METHODS: Peripheral blood mononuclear cells (PBMCs) obtained from allergic individuals and non-allergic controls, either during the pollen season or out of season, were stimulated with either TG extract or a pool of previously identified immunodominant antigenic regions. RESULTS: PBMCs from allergic subjects exhibit higher IL-5 and IL-10 responses in season than when collected out of season. In the case of non-allergic subjects, as expected we observed lower IL-5 responses and robust production of IFN-γ compared to allergic individuals. Strikingly, non-allergic donors exhibited an opposing pattern, with decreased immune reactivity in season. The broad down-regulation in non-allergic donors indicates that healthy individuals are not oblivious to allergen exposure, but rather react with an active modulation of responses following the antigenic stimulus provided during the pollen season. Transcriptomic analysis of allergen-specific T cells defined genes modulated in concomitance with the allergen exposure and inhibition of responses in non-allergic donors. CONCLUSION AND CLINICAL RELEVANCE: Magnitude and functionality of T helper cell responses differ substantially in season vs. out of season in allergic and non-allergic subjects. The results indicate the specific and opposing modulation of immune responses following the antigenic stimulation during the pollen season. This seasonal modulation reflects the enactment of specific molecular programmes associated with health and allergic disease.

Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

BACKGROUND: Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity among grass-pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. OBJECTIVES: We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4(+) T cell level among DR04:01 restricted Pooideae grass-pollen T-cell epitopes. METHODS: After in vitro culture of blood mono-nucleated cells from grass-pollen-allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labelled DR04:01/Phleum pratense tetramers and PE-labelled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity among allergen-specific DR04:01-restricted T-cells in six subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass-pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. RESULTS: T-cells with various degrees of cross-reactive profiles could be detected. Poa p 1 97-116 , Lol p 1 221-240 , Lol p 5a 199-218 , and Poa p 5a 199-218 were identified as minimally cross-reactive T-cell epitopes that do not show cross-reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally cross-reactive T-cell epitopes are present in Grass-pollen-allergic subjects. CONCLUSIONS: Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross-reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells suggest that a multiple allergen system should be considered for immunotherapy instead of a mono-allergen system.

Allergen-specific CD4+ T cell responses in peripheral blood do not predict the early onset of clinical efficacy during grass pollen sublingual immunotherapy.

BACKGROUND: Surrogate biomarkers of efficacy are needed in support of allergen-specific immunotherapy. OBJECTIVE: The aim of this study was to relate changes in peripheral CD4(+) T cell responses to clinical efficacy during sublingual immunotherapy (SLIT). METHODS: Allergen-specific CD4(+) T cell responses were assessed in peripheral blood mononuclear cells (PBMCs) from 89 grass pollen-allergic individuals enrolled in a double-blind placebo-controlled SLIT study conducted in an allergen exposure chamber (ClinicalTrials.gov NCT00619827). Surface phenotype, proliferative responses, cytokine production and gene expression were analysed in coded samples at baseline, and after 2 and 4 months of SLIT, in PBMCs after in vitro allergen stimulation or among MHC class II/peptide (pMHCII)-tetramer-positive CD4(+) T cells. RESULTS: SLIT induced a 29.3% improvement of the average rhinoconjunctivitis total symptom score in the active group, when compared to the placebo group. In parallel, only minor changes in proportions of CD4(+) T cells expressing Th1 (CCR5(+), CXCR3(+)), Th2 (CRTh2(+), CCR4(+)) and Treg (CD25(+), CD127(-), Foxp3(+)) markers were detected. A down-regulation of IL-4 and IL-10 gene expression and IL-10 secretion (P < 0.001) were observed, as well as a decrease in the frequency of potential "pro-allergic" CD27(-) Th2 cells from patients receiving active tablets (P < 0.001), but without any correlation with clinical benefit. pMHCII-tetramer analyses failed to document any major impact in both numbers and polarization of circulating Phl p 1- and Phl p 5-specific CD4(+) T cells, confirming that early clinical improvement during SLIT is not associated with dramatic alterations in T lymphocyte responses. CONCLUSION & CLINICAL RELEVANCE: Changes in patterns of peripheral CD4(+) T cells are not markers for the early onset of efficacy during SLIT.