Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Binda, Elisa; Erhart, Dominik; Schenk, Mirjam; Zufferey, Christel; Renzulli, Pietro; Mueller, Christoph

doi:10.1016/j.jim.2009.02.006

Cited by 11 publications

(7 citation statements)

References 27 publications

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“…Small intestinal IEL were isolated as described previously 33 . Briefly, tissues were placed in Ca 2+ and Mg 2+ ‐free Hanks’ balanced salt solution (HBSS) containing 10 m m HEPES and 2% horse serum.…”

Section: Methodsmentioning

confidence: 99%

Production of interferon‐γ by activated T‐cell receptor‐αβ CD8αβ intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

et al. 2009

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Summary Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T‐cell receptor (TCR)‐γδ subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR‐αβ CD8αβ and TCR‐αβ CD8αα subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt‐derived cell‐line mICcl2 and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate‐conjugated (FITC‐) dextran. The TCR‐αβ CD8αα IEL and resting TCR‐αβ CD8αβ IEL did not affect TER of mICcl2 or flux of FITC‐dextran. In contrast, activated TCR‐αβ CD8αβ IEL clearly disrupted the integrity of the mICcl2 monolayer. No disrupting effect was seen with activated TCR‐αβ CD8αβ IEL from interferon‐γ knockout mice. These findings demonstrate that secretion of interferon‐γ by activated TCR‐αβ CD8αβ IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

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Section: Methodsmentioning

confidence: 99%

Production of interferon‐γ by activated T‐cell receptor‐αβ CD8αβ intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

et al. 2009

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“…In addition, they express a range of other activating and inhibitory receptors, such as NK receptors, allowing them to rapidly respond to danger and stress signals in their microenvironment [1]. Further, as resident T cells, IEL do not recirculate and are retained in the epithelium by interactions such as αEβ7 (also known as CD103) on their surface binding to E-cadherin on epithelial cells [2]. However, there is still relatively limited research into the functionality of these unconventional T cells, compared to T cell found in the spleen and blood.…”

Section: Introductionmentioning

confidence: 99%

Isolation, Characterization, and Culture of Intestinal Intraepithelial Lymphocytes

James

Vandereyken

Swamy

2020

Methods in Molecular Biology

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Intestinal intraepithelial lymphocytes (IEL) comprise distinct groups of innate-like and memory T cells that collectively form one of the largest T cell compartments in the body. IEL are located within the intestinal epithelium and are the first immune cells in the gut to interact with the food, microbiota, and pathogens that the gut is continually exposed to. IEL can respond rapidly to external insults to protect the small intestinal epithelium but are also considered regulatory cells that are important to maintain the homeostasis of the gut. However, the mechanisms of IEL activation and their interactions with the epithelium remain largely elusive. Indeed, IEL are not commonly evaluated even in studies of gut immunology, potentially because they are perceived as being difficult to isolate and study. In this protocol, we present a simplified method to isolate IEL from the murine small intestine and provide representative data for flow cytometric analyses of the different IEL subsets. We also outline two procedures for culturing IEL, which can permit functional studies and coculture with epithelial cells. These strategies should make studies of this large but enigmatic T cell compartment more accessible and open up understanding of homeostatic mechanisms in the intestine, and tissue-associated immunity.

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“…In contrast to our method, elutriation appears to be more interesting with respect to short-term cultivations (Willheim et al 1995). Hence, the disinfection of elutriation rotor is still carried out simply by a less reliable ethanol (Binda et al 2009). Nevertheless, the possibility to remove impurities of variously differentiated cells by magnetic separation (Willheim et al 1995) appears to be an interesting supplement to our future method improvement.…”

Section: Comparison Of Our Lmc Separation With Related Methodsmentioning

confidence: 93%

Isolation of Rat Lung Mast Cells for Purposes of One-Week Cultivation Using Novel Percoll Variant Percoll PLUS

Kubrycht

Maxová²,

Nyč³

et al. 2011

Physiol Res

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Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative “asymptotically isotonic” stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasting cultivation and observed some model morphological changes of LMC.

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Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Cited by 11 publications

References 27 publications

Production of interferon‐γ by activated T‐cell receptor‐αβ CD8αβ intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Production of interferon‐γ by activated T‐cell receptor‐αβ CD8αβ intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Isolation, Characterization, and Culture of Intestinal Intraepithelial Lymphocytes

Isolation of Rat Lung Mast Cells for Purposes of One-Week Cultivation Using Novel Percoll Variant Percoll PLUS

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