The influence of different stimulation conditions on the assessment of antigen‐induced CD154 expression on CD4<sup>+</sup> T cells

Meier, Stefan; Stark, Regina; Frentsch, Marco; Thiel, Andreas

doi:10.1002/cyto.a.20640

Cited by 38 publications

(27 citation statements)

References 22 publications

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“…However, little is known about the underlying mechanisms and the function of Th cells induced by oral antigen. In this study, low-dose oral KLH induced an antigen-specific systemic CD4 The frequencies of KLH-specific CD4 1 T cells as identified by the expression of CD154 after short-term in vitro stimulation [20,24] were low but similar to the frequencies of antigen- Figure 3. Oral KLH results in a shift of functional subpopulations of KLH-specific Th cells induced by subsequent parenteral immunization.…”

Section: Discussionsupporting

confidence: 49%

“…The frequencies of KLH-specific CD4 1 T cells as identified by the expression of CD154 after short-term in vitro stimulation [20,24] were low but similar to the frequencies of antigen- MLN DC originating from the small intestinal lamina propria [31]. Similar mechanisms could explain the predominant expression of the skin-homing receptor CLA and the gut-homing receptor integrin b7 on KLH-specific Th cells after parenteral and oral immunization, respectively, in our study and earlier human reports [13,[32][33][34][35][36].…”

Section: Discussionmentioning

confidence: 99%

“…1). According to the earlier studies [20,24] a threefold increase above background was selected as cut-off point. For proliferation analysis, the CellTrace TM CFSE Cell Proliferation kit (Invitrogen, Karlsruhe, Germany) was used according to the manufacturer's instructions.…”

Section: Cell Isolation and Stimulationmentioning

confidence: 99%

“…In this study, low-dose oral KLH induced an antigen-specific systemic CD4 1 T-cell response with a shift toward a Th type 2 cytokine pattern. In a subsequent systemic immune response induced by parenteral immunization, low-dose oral KLH amplified B-cell responses and, most strikingly, shifted the cytokine pattern of KLH-specific CD4 1 T cells from a Th type 1 toward a Th type 2, suggesting a novel effect of oral immunomodulation.The frequencies of KLH-specific CD4 1 T cells as identified by the expression of CD154 after short-term in vitro stimulation [20,24] were low but similar to the frequencies of antigen- MLN DC originating from the small intestinal lamina propria [31]. Similar mechanisms could explain the predominant expression of the skin-homing receptor CLA and the gut-homing receptor integrin b7 on KLH-specific Th cells after parenteral and oral immunization, respectively, in our study and earlier human reports [13,[32][33][34][35][36].…”

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confidence: 99%

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Modulation of systemic antigen‐specific immune responses by oral antigen in humans

et al. 2010

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Oral antigen uptake can induce systemic immune responses ranging from tolerance to immunity. However, the underlying mechanisms are poorly understood, especially in humans. Here, keyhole limpet hemocyanin (KLH), a neoantigen which has been used in earlier studies of oral tolerance, was fed in a repeated low-dose and a single high-dose protocol to healthy volunteers. KLH-specific CD4 1 T-cell proliferation and cytokine production, as well as KLH-specific serum Ab and the effects of oral KLH on a subsequent parenterally induced systemic immune response, were analyzed. Repeated low-dose oral KLH alone induced antigen-specific CD4 1 T cells positive predominantly for the gut-homing receptor integrin b7 and the cytokines IL-2 and TNF-a; some CD4 1 T cells also produced IL-4.Oral feeding of KLH accelerated a subsequent parenterally induced systemic CD4 1 T-cell response. The cytokine pattern of KLH-specific CD4 1 T cells shifted toward more IL-4-and IL-10-and less IFN-c-, IL-2-and TNF-a-producing cells. The parenterally induced systemic KLH-specific B-cell response was accelerated and amplified by oral KLH. The impact of single high-dose oral KLH on antigen-specific immune responses was less pronounced compared with repeated low-dose oral KLH. These findings suggest that oral antigen can effectively modulate subsequently induced systemic antigen-specific immune responses. Immunomodulation by oral antigen may offer new therapeutic strategies for Th type1-mediated inflammatory diseases and for the development of vaccination strategies. [1,[4][5][6][7]. Systemic tolerance in these models may result from a variety of effects including clonal anergy, clonal deletion and active regulation [2]. Understanding the mechanisms leading to such diverse outcomes after mucosal antigen uptake is not only important for mucosal vaccination [3,7], but may also offer new options for immunomodulation or the treatment of autoimmune diseases [8][9][10]. Unfortunately, responses to oral antigen differ significantly between species [8,11], and human studies are rare.Similar to mouse models, systemic immune responses can be induced in humans by oral uptake of pathogens which invade the mucosa, e.g. Salmonella typhi Ty21a [12,13], or of toxins which can bind to mucosal epithelial cells and compromise the mucosal barrier, e.g. cholera toxin [14,15].The induction of human oral tolerance has been described [16][17][18] after feeding keyhole limpet hemocyanin (KLH), a neoantigen which is safe to use in humans. Compared with rodent studies, the antigen doses used were much lower and effects observed were not as uniform: reduced delayed-type hypersensitivity (DTH) [17,18] and antigen-specific proliferation of PBMC [16][17][18] have been reported after feeding KLH, although KLH-specific B-cell responses were unaffected [16] or even amplified [17,18].Functional Th-cell subsets are believed to determine the type of immune responses, but have not been examined after oral antigen challenge in humans. The frequencies of human antigenspecific Th cells or e...

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Section: Discussionsupporting

confidence: 49%

Section: Discussionmentioning

confidence: 99%

Section: Cell Isolation and Stimulationmentioning

confidence: 99%

mentioning

confidence: 99%

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Modulation of systemic antigen‐specific immune responses by oral antigen in humans

et al. 2010

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“…The sensitivity and range of applications of the cytokine secretion assay is enhanced by inclusion of activation markers, such as CD154 (32), CD137 (33), or CD161 for Tregs (34), or antigen-receptors of T-lymphocytes by MHC multimer technologies (26,35). For MACS and FACS detection of antigen-reactive T lymphocytes, CD154 for antigen-specific CD4 cells and CD137 for antigen-specific CD8 cells have been developed, allowing to assess the entity of specific cells, irrespective of their histocompatibility-restriction or peptide specificity (32,33,36,37). Antigen-specific T lymphocytes have been used for ex vivo analyses, and when isolated by MACS, are just about to enter clinical settings, for example, to transfer virus protection to incompetent patients (24,38,39).…”

Section: Review Articlementioning

confidence: 99%

Small but mighty: How the MACS^®‐technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years

Grützkau¹,

Radbruch²

2010

Cytometry Pt A

127

109

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Today, magnetic cell sorting and flow cytometric cell sorting both are state-of-the-art technologies, with a plethora of applications in biology and biomedicine. Both technologies have their stand-alone applications, but they also perfectly complement each other, in particular for the analysis and isolation of fragile and rare cells. The technological evolution from simple magnets and steel wool separation columns to sophisticated instrumentation and automated procedures has paved the way for magnetic cell separation in the scientific community. This review will focus on applications of magnetic cell sorting with commercially available paramagnetic MACS 1 -nanoparticles (Miltenyi Biotech, Bergisch-Gladbach, Germany) that have allowed unprecedented approaches to the exploration of the immune system in basal and clinical research of humans and rodents. Thus, during the last 20 years this technology has been continuously developed so that the widest array of leukocyte subsets, stem cells, and connective tissue cells can be addressed by MACS 1 -conjugated antibodies directed against cell-specific surface antigens or secreted cytokines for research and clinical applications. ' 2010 International Society for Advancement of Cytometry

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Detection of Intracellular Cytokines by Flow Cytometry

2015

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Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.

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The influence of different stimulation conditions on the assessment of antigen‐induced CD154 expression on CD4⁺ T cells

Cited by 38 publications

References 22 publications

Modulation of systemic antigen‐specific immune responses by oral antigen in humans

Modulation of systemic antigen‐specific immune responses by oral antigen in humans

Small but mighty: How the MACS^®‐technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years

Detection of Intracellular Cytokines by Flow Cytometry

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The influence of different stimulation conditions on the assessment of antigen‐induced CD154 expression on CD4+ T cells

Cited by 38 publications

References 22 publications

Modulation of systemic antigen‐specific immune responses by oral antigen in humans

Modulation of systemic antigen‐specific immune responses by oral antigen in humans

Small but mighty: How the MACS®‐technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years

Detection of Intracellular Cytokines by Flow Cytometry

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Product

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The influence of different stimulation conditions on the assessment of antigen‐induced CD154 expression on CD4⁺ T cells

Small but mighty: How the MACS^®‐technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years