purification and analysis of mice naive cd4+ t-cells using magnetic bead-based negative selection

<meta charset="utf8"></head><body>Diferenciação de células Th17 de células T CD4 + virgens usando citometria de fluxo

After leaving the thymus, naive CD4+ T lymphocytes pass through secondary lymphoid organs. Antigen-presenting cells that transmit homologous antigens to na&#239;ve CD4+ T cells activate them, starting a series of differentiation programs that eventually result in the production of highly specialized T helper (Th) cell lineages1. Interleukin 17 (IL-17) production characterizes Th17 cells, a subpopulation of pro-inflammatory Th cells2. Th17 cells play a role in the host&#39;s defense against extracellular pathogens and in the pathogenesis of many autoimmune diseases, such as autoimmune uveitis and multiple sclerosis. Signals from T-cell receptors and cytokines IL-6 and transforming growth factor-&#946; (TGF-&#946;) induce the differentiation of naive T cells into Th17 cells through the phosphorylation of signal transducer and activator of transcription (STAT)33. STAT3 is further amplified in a positive feedback loop through signaling mediated by IL-23 and IL-214,5. Phosphorylation of STAT3 can induce the expression of the transcription factors ROR&#947;t and ROR&#945;, which act as master switches regulating the cytokine profile of IL-17A, IL-17F, IL-21, and IL-22 in Th17 cells6. However, it has been reported that IL-6- and TGF-&#946;-induced Th17 cells are insufficient to trigger autoimmune diseases, which require co-stimulation by IL-23 or separate co-stimulation of IL-6, IL-1&#946;, and IL-23 in the absence of TGF-&#946;7,8.
Th17 subsets that cannot effectively induce experimental autoimmune encephalomyelitis (EAE) are sometimes referred to as non-pathogenic Th17 while Th17 subsets that can induce EAE are known as pathogenic Th179. Current studies have shown that although pathogenic Th17 and non-pathogenic Th17 co-express the core transcription factor ROR&#947;t, there are great differences in the ability to produce IL-17A and pro-inflammatory and anti-inflammatory properties10. In addition to the high expression of ROR&#947;t, CCR6, STAT4, and RUNX4, which are common characteristic transcription factors of Th17, pathogenic Th17 cells also show additional gene signal expression characteristics related to the disease, such as TBX21, IFN-&#947;, and CXCR3, which have the characteristics of Th1 cell subsets. Pathogenic Th17 cells can secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-&#947;, TNF-&#945;, and other cytokines11,12. The phenotype of non-pathogenic Th17 cells is unstable, and only under the stimulation of CD3 and cytokine IL-2 can some of these cells differentiate into pathogenic Th17 cells. Therefore, in common clinical disease models such as rheumatoid arthritis, multiple sclerosis, and acute respiratory distress syndrome, pathogenic Th17 cells primarily exert pathogenic effects.
Pathogenic and non-pathogenic Th17 cells can differentiate in vitro under the influence of different cytokines. In recent years, several studies have proposed methods for inducing the differentiation of Th17 cells using different types and concentrations of cytokines. Th17 cells are stimulated by a combination of IL-6, IL-1&#946;, and IL-2313,14,15,16. It has been proved that IL-6 and TGF-&#946;, two cytokines necessary for Th17 cell differentiation, synergistically regulate the expression of ROR&#947;t and Th17 cell differentiation by interacting with two different conserved non-coding DNA sequences at the Rorc gene locus17. The stable phase of pathogenic Th17 cells is mainly maintained by IL-2318,19. IL-23 binds to its receptor and activates the JAK-STAT signaling pathway20, thereby causing phosphorylation of Jak2 and Tyk2 and promoting phosphorylation of STAT1, STAT3, STAT4, and STAT5. IL-4 and IFN-&#947; are negative regulators of this pathway. However, studies have shown that IL-1&#946; may positively regulate the transcription of Ror&#945; and Ror&#947;t through the mTOR pathway to maintain the stability of the Th17 cell phenotype21.
Due to the heterogeneity of numerous studies, we chose the induction protocols for pathogenic and non-pathogenic Th17 cells from the latest research as controls22. The results indicate that, assuming that everything is carried out according to this protocol, after 5 days of culture under the condition of generating pathogenic Th17, more than 90% of the surviving cells can be pathogenic Th17 cells.

<meta charset="utf8"></head><body>Autor em destaque: Alcançando diferenciação in vitro de alta pureza de células Th17 usando modulação de concentração de citocinas

Diferenciação in vitro de células T CD4+ virgens em células Th17 patogênicas em camundongos

Purificação e análise de células T CD4+ virgens de camundongos usando seleção negativa baseada em esferas magnéticas

purification-analysis-mice-naive-cd4-t-cells-using-magnetic-bead

Research

JoVE Journal

Immunology and Infection

Purification and Analysis of Mice Naive CD4+ T-Cells Using Magnetic Bead-Based Negative Selection

314 visualizações. Para começar, prepare uma suspensão de célula única esplênica de camundongo com um vezes 10 elevado a oito células por mililitro em um volume de 0,1 a dois mililitros. Adicione 50 microlitros de soro de rato à amostra. Transfira a amostra para um tubo inferior redondo de poliestireno de cinco mililitros.Adicione 50 microlitros de coquetel de isolamento à amostra. Misture bem e incube por 7,5 minutos em temperatura ambiente. Agora adicione 50 mi...

Vídeo: Purificação e análise de células T CD4+ virgens de camundongos usando seleção negativa baseada em esferas magnéticas

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...