Flow cytometric analysis of cytokine expression in short-term allergen-stimulated T cells mirrors the phenotype of proliferating T cells in long-term cultures

Hemelen, Dries Van; Elberink, Joanne N.G. Oude; Bohle, Barbara; Heimweg, Janneke; Nawijn, Martijn C.; Oosterhout, Antoon J. M. van

doi:10.1016/j.jim.2011.06.019

Cited by 9 publications

(9 citation statements)

References 27 publications

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“…Confirmation of a strong correlation between Th2 cells responding to cat dander and levels of cat-dander IgE in this paper provides further support, in addition to confirmation of phenotypic data established using MHC class II tetramers. In keeping with our previous studies of responses to birch allergen [3] and other studies [7,8,11], we found that responses to cat and grass allergens were very mixed, with activated Th2, Th1 and Tr1-like lymphocytes identified in varying proportions in allergic and non-allergic participants. Although the frequency of Th2 cells was significantly higher amongst allergic participants compared to non-allergic controls, there was considerable overlap, and the frequency of Th1 cells did not differ significantly.…”

Section: Discussionsupporting

confidence: 91%

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Characterisation of CD154+ T cells following ex vivo allergen stimulation illustrates distinct T cell responses to seasonal and perennial allergens in allergic and non-allergic individuals

et al. 2013

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BackgroundAllergic sensitisation has been ascribed to a dysregulated relationship between allergen-specific Th1, Th2 and regulatory T cells. We sought to utilise our short-term CD154 detection method to further analyse the relationship between these T cell subsets and investigate differences between seasonal and perennial allergens. Using peripheral blood samples from grass-allergic, cat-allergic and healthy non-atopic subjects, we compared the frequencies and phenotype of CD154-positive T helper cells following stimulation with seasonal (grass) and perennial (cat dander) allergens.ResultsWe identified a higher frequency of CD154+ T cells in grass-allergic individuals compared to healthy controls; this difference was not evident following stimulation with cat allergen. Activated Th1, Th2 and Tr1-like cells, that co-express IFNγ, IL4 and IL10, respectively, were identified in varying proportions in grass-allergic, cat-allergic and non-allergic individuals. We confirmed a close correlation between Th1, Th2 and Tr1-like cell frequency in non-allergic volunteers, such that the three parameters increased together to maintain a low Th2: Th1 ratio. This relationship was dysregulated in grass-allergic individuals with no correlation between the T cell subsets and a higher Th2: Th1 ratio. We confirmed previous reports of a late-differentiated T cell phenotype in response to seasonal allergens compared to early-differentiated T cell responses to perennial allergens.ConclusionsThe findings confirm our existing work illustrating an important balance between Th1, Th2 and Tr1-like responses to allergens in health, where Th2 responses are frequently observed, but balanced by Th1 and regulatory responses. We confirm previous tetramer-based reports of phenotypic differences in T cells responding to seasonal and perennial allergens.

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Section: Discussionsupporting

confidence: 91%

“…A previous study with CD154/tetramer co-staining after prolonged cell culture followed by re-stimulation questioned the validity of CD154 as a marker for allergen-specific T cells (ref). However, these conditions are known to invalidate the assay, which is only reliable after short-term stimulation [11]. …”

Section: Discussionmentioning

confidence: 99%

Characterisation of CD154+ T cells following ex vivo allergen stimulation illustrates distinct T cell responses to seasonal and perennial allergens in allergic and non-allergic individuals

et al. 2013

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“…IFNγ from NK cells, Th1 and cytotoxic T cells and IL13 from Th2 cells, but also group 2 innate lymphoid cells including natural helper cells and nuocytes [39]. The parallel induction of a Th1 as well as a Th2 response is a common phenomenon [27], [40], [41]. Moreover, in allergics the ratio between Th1/Th2 cells seems rather to reflect the allergic status than the presence of Th1 cells alone [41].…”

Section: Discussionmentioning

confidence: 99%

The Major Cow Milk Allergen Bos d 5 Manipulates T-Helper Cells Depending on Its Load with Siderophore-Bound Iron

et al. 2014

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The mechanisms of allergic sensitization to milk are still elusive. The major allergen Bos d 5 belongs to the lipocalin-family and thus is able to transport numerous ligands. In this study we investigated its ability to bind to iron-siderophore complexes and tested the immune-modulatory properties of Bos d 5 in either forms. Structural and in silico docking analysis of Bos d 5 revealed that Bos d 5 is able to bind to iron via catechol-based flavonoids (quercetin, myricetin, luteolin) that act as siderophores as confirmed by spectral-analysis and iron staining. Calculated dissociation constants of docking analyses were below 1 µM by virtual addition of iron. When incubated with human peripheral blood mononuclear cells (PBMCs), only the apo-form of Bos d 5 led to an increase of CD4+positive cells and significantly elevated IL13 and IFNγ-levels. In contrast, holo-Bos d 5 decreased numbers of CD4 expressing cells and induced apoptosis. Taken together, our data give evidence that Bos d 5 is capable of binding iron via siderophores. Moreover, our data support for the first time the notion that the form of application (apo- or holo-form) is decisive for the subsequent immune response. The apo-form promotes Th2 cells and inflammation, whereas the holo-form appears to be immunosuppressive.

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“…A previous study with CD154/ tetramer co-staining after prolonged cell culture and re-stimulation questioned the validity of CD154 as a marker for allergen-specific T cells [10], but these experimental conditions are known to invalidate the assay, which is only reliable after short-term stimulation [6]. The close relationship between Th1, Th2 and Tr1-like cells was first reported by Akdis et al [2] using cytokine capture technology.…”

Section: Discussionmentioning

confidence: 99%

“…The method was shown to produce similar results compared to cultured T cell systems following allergen stimulation [6], and has been used by Campbell et al to track T cell responses during experimental ragweed desensitisation [7]. However, detailed phenotyping of CD154 + T cells after allergen stimulation has not been reported to date.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of CD154+ T cells following ex vivo birch allergen stimulation defines a close relationship between T cell subsets in healthy volunteers

et al. 2013

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BackgroundAllergic sensitisation has been ascribed to a dysregulated relationship between allergen-specific Th1, Th2 and regulatory T cells. We hypothesised that the relationship between these T cell subsets could be better defined using a short-term allergen stimulation system followed by direct analysis of CD154-positive T cells. Using peripheral blood samples from birch pollinosis patients and healthy non-atopic controls, we sought to explore the frequencies and phenotype of birch-stimulated CD154-positive T helper cells following ex vivo birch allergen stimulation.ResultsActivated CD154-positive Th1, Th2 and Tr1-like cells, that co-expressed IFNγ, IL-4 and IL-10 respectively, were identified in both birch-allergic and non-allergic participants. We observed a close correlation between Th1, Th2 and Tr1-like cell frequency in non-allergic volunteers, such that the three parameters increased together to maintain a low Th2: Th1 ratio. The relationship between Th1, Th2 and Tr1-like responses was dysregulated in birch-allergic patients, with abrogation of the IL-10 response and a higher Th2: Th1 ratio. A close correlation was observed between Th2 cell frequency and the absolute concentration of birch-specific IgE within the birch-allergic group, and we confirmed previous reports of a more differentiated T cell phenotype in allergic subjects.ConclusionsThe findings demonstrate an important balance between IFNγ, IL-4 and IL-10 T cell responses to birch allergen in health, where Th2 responses to allergens were frequently observed, but apparently balanced by Th1 and regulatory responses. The detection of CD154 positive T cells after short-term antigen stimulation may be a useful method for the detection of T cell responses to allergens when cost, speed and convenience are priorities.

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Flow cytometric analysis of cytokine expression in short-term allergen-stimulated T cells mirrors the phenotype of proliferating T cells in long-term cultures

Cited by 9 publications

References 27 publications

Characterisation of CD154+ T cells following ex vivo allergen stimulation illustrates distinct T cell responses to seasonal and perennial allergens in allergic and non-allergic individuals

Characterisation of CD154+ T cells following ex vivo allergen stimulation illustrates distinct T cell responses to seasonal and perennial allergens in allergic and non-allergic individuals

The Major Cow Milk Allergen Bos d 5 Manipulates T-Helper Cells Depending on Its Load with Siderophore-Bound Iron

Characterisation of CD154+ T cells following ex vivo birch allergen stimulation defines a close relationship between T cell subsets in healthy volunteers

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