Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Xing, Yan; Hogquist, Kristin A.

doi:10.3791/51780

Cited by 41 publications

(35 citation statements)

References 37 publications

(62 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…19 Human TECs were prepared by Liberase-TH (Roche life sciences) digestion, as described previously. 20 Flow cytometry and FACS analysis CD4 + CD8 − CD25 + Foxp3 − , CD4 + CD8 − CD25 − Foxp3 low and CD4 + CD8 − CD25 + Foxp3 + constitute o1% of total thymocytes. Therefore, CD4SP thymocytes were purified using an EasySep mouse CD4 + T-cell isolation kit to enrich these populations.…”

Section: Thymic Organ Culture and Thymic Epithelial Cell Isolationmentioning

confidence: 99%

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

et al. 2018

View full text Add to dashboard Cite

Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40 mice had significantly reduced numbers of CD25Foxp3 tTreg precursors and CD25Foxp3 mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.Cellular and Molecular Immunology advance online publication, 26 March 2018; doi:10.1038/cmi.2018.8.

show abstract

Section: Thymic Organ Culture and Thymic Epithelial Cell Isolationmentioning

confidence: 99%

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Their rarity (~200,000 per mouse thymus) and fragility made it a challenging task to isolate TECs. The recent utilization of highly purified collagenase in TEC isolation have significantly increased the numbers of live cells following the enzymatic digestion 15–19 .…”

Section: Discussionmentioning

confidence: 99%

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Tajima

Liu

Pradhan

et al. 2016

JoVE

View full text Add to dashboard Cite

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

show abstract

“…Protocol was carried out as previously described (16). Briefly, thymus lobes were cut in small pieces and cleaned for blood and connective tissue in RPMI (Lonza) supplemented with 10% FCS, 2 mM L-glutamine and 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we investigated the effects of CV-B4 E2 infection on Igf2 transcription using an enrichment method of TECs (15, 16) from an outbred susceptible mice strain (9). We also explored mechanisms of the regulation of Igf2 transcription in MTE4-14 cells induced by a short CV-B4 infection.…”

Section: Introductionmentioning

confidence: 99%

Mechanisms ofIgf2inhibition in thymic epithelial cells infected by coxsackievirus CV-B4

Michaux

Halouani

Trussart

et al. 2020

Preprint

View full text Add to dashboard Cite

Epidemiological studies have evidenced a link between type 1 diabetes (T1D) and infections by enteroviruses, especially with coxsackievirus B4 (CV-B4). CV-B4 is able to infect human and murine thymic epithelial cells (TECs) and, in a murine TEC line, we have shown that the diabetogenic strain CV-B4 E2 decreases transcription of insulin-like growth factor 2 gene (Igf2), coding for the self-peptide of the insulin family. Here we show that in CV-B4 infection of mice alters Igf2 transcripts isoforms in TECs, followed by a decrease of pro-IGF2 precursor in the thymus. CV-B4 infection of a murine TEC line decreases Igf2 P3 promoter activity by targeting the region −68 to −22 upstream of the transcription start site (TSS) whereas Igf2 transcripts stability is not affected, pointing towards a regulation of Igf2 transcription. Our data also show that CV-B4 decreases IL-6/STAT3 signaling in vitro. This study provides new knowledge about the regulation of intrathymic Igf2 transcription by CV-B4 and reinforces the hypothesis that CV-B4 infection of the thymus could break central self-tolerance of the insulin family by decreasing Igf2 transcription and IGF2 presentation in thymus epithelium.IMPORTANCECoxsackievirus B4 represents one of the most important environmental factors associated to type 1 diabetes, autoimmune disease for which no curative treatment exist. The diabetogenic strain Coxsackievirus B4 E2 was previously shown to decrease Igf2 expression, important player for central tolerance towards insulin, in a thymic epithelial cell line. The understanding of Igf2 regulation mechanisms during coxsackievirus B4 infection represents an interest for the understanding of central tolerance development but also for Igf2 transcriptional regulation itself, still poorly understood.Here we demonstrate that, some transcripts isoforms of Igf2 are also decreased in thymic epithelial cells in vivo. Moreover, we show that this decrease is induced by an alteration of specific regions of Igf2 P3 promoter and may be linked by a decrease of STAT3 signaling. In fine we hope that this work could lead to future therapies leading to reprogramming central tolerance towards β cells antigens via Igf2 expression.

show abstract

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Cited by 41 publications

References 37 publications

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

Critical role of OX40 signaling in the TCR-independent phase of human and murine thymic Treg generation

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Mechanisms ofIgf2inhibition in thymic epithelial cells infected by coxsackievirus CV-B4

Contact Info

Product

Resources

About