lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and <em>Ex Vivo</em> Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Radboud university medical centre

T lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.

Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling.

Regulatory T-cells (Treg) are crucial for immune homeostasis and prevention of immune pathology. Yet, Treg may lose Foxp3 and start secreting IL-17, dependent on environmental cues. Our previous data revealed that Treg from severe psoriasis patients are particularly prone to such conversion. The question of how to maintain Treg stability in the context of inflammation awaits immediate resolution. The pan-protein kinase C (PKC) inhibitor sotrastaurin has shown efficacy in clinical trials of psoriasis. Here, we show that sotrastaurin inhibited effector T-cell responses, whereas the regulatory response was enhanced. Sotrastaurin prevented TCR/CD28-induced T-cell activation and pro-inflammatory cytokine production, but preserved a stable Treg phenotype as evidenced by maintenance of suppressive capacity, high Foxp3 and CD25 expression, and lack of IL-17A and IFNγ production. Moreover, in both circulating and dermal psoriatic Treg, prone to rapid induction of IL-17, sotrastaurin enhanced Foxp3 expression and prevented IL-17A and IFNγ production even when stimulated in the presence of the helper T 17-enhancing cytokines IL-1β or IL-23. Thus, pharmacological inhibition of PKC may serve as a powerful tool to concurrently inhibit effector T cells and to facilitate Treg, thereby showing therapeutic potential for the treatment of psoriasis.

Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.

Self-tolerance and immune homeostasis are orchestrated by FOXP3(+) regulatory T cells (Treg). Recent data have revealed that, upon stimulation, Treg may exhibit plasticity towards a pro-inflammatory phenotype, producing IL-17 and/or IFN-γ. Such deregulation of Treg may contribute to the perpetuation of inflammatory processes, including graft-versus-host-disease. Thus, it is important to identify immunomodulatory factors influencing Treg stability. Platelet-derived microparticles (PMPs) are involved in hemostasis, vascular health, and have recently been shown to be intimately involved in (pathogenic) immune responses. Therefore, we investigated whether PMPs have the ability to affect Treg plasticity. PMPs were co-cultured with healthy donor peripheral blood-derived Treg that were stimulated with anti-CD3/CD28 monoclonal antibodies in the presence of IL-2, IL-15 and IL-1β. PMPs prevented the differentiation of peripheral blood-derived Treg into IL-17 and IFN-γ-producing cells, even in the presence of the IL-17 driving pro-inflammatory cytokine IL-1β. The mechanism of action by which PMPs prevent Treg plasticity consisted of rapid and selective P-selectin-dependent binding of PMPs to a CCR6(+)HLA-DR(+) memory-like Treg subset, and their ability to inhibit Treg proliferation, in part through CXCR3 engagement. The finding that around 8% of Treg in the circulation of healthy individuals are CD41(+)P-selectin(+) and that distinct binding of patient plasma PMPs to Treg was observed supports in vivo relevance. These findings open the exciting possibility that PMPs actively regulate the immune response at sites of (vascular) inflammation, where they are known to accumulate and interact with leukocytes, consolidating the (vascular) healing process.

Article	Year
Molecular pathway profiling of T lymphocyte signal transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-mediated signaling. BMC immunology\| PubMed ID: 22413885	2012
Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production. The Journal of investigative dermatology\| PubMed ID: 24192715	2014
Platelet-derived microparticles inhibit IL-17 production by regulatory T cells through P-selectin. Blood\| PubMed ID: 26903549	2016

Xuehui He

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