RESUMO
The gastrointestinal immune system plays a pivotal role in the host relationship with food antigens, the homeostatic microbiome and enteric pathogens. Here, we describe how to collect and process liver and intestinal samples to efficiently isolate and analyse resident immune cells. Furthermore, we describe a step-by-step methodology showing how to high-dimensionally immunophenotype resident leucocytes using cytometry by time-of-flight, providing a well-characterized antibody platform that allows the identification of every leucocyte subset simultaneously. This protocol also includes instructions to purify and cultivate primary murine hepatocytes, a powerful tool to assess basic cell biology and toxicology assays. Gut and liver samples from the same mouse can be collected, processed and stained in less than 6 hr. This protocol enables the recovery of several populations of purified and viable immune cells from solid and fibrous organs, preventing unwanted loss of adherent cells during isolation.
Assuntos
Imunofenotipagem/métodos , Mucosa Intestinal/citologia , Leucócitos/citologia , Fígado/citologia , Linfonodos/citologia , Animais , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Citometria de Fluxo , Mucosa Intestinal/imunologia , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BLAssuntos
Citometria de Fluxo/tendências , Análise de Célula Única/métodos , Aptâmeros de Nucleotídeos , Biomarcadores , Bases de Dados como Assunto , Citometria de Fluxo/instrumentação , Citometria de Fluxo/normas , Genômica , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Microbiota/genética , Microscopia de Fluorescência , Reprodutibilidade dos Testes , Software , Estudos de Validação como AssuntoRESUMO
CD4+ regulatory T cells (CD4Tregs) play a critical role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. IL-2 supports the proliferation and survival of CD4Tregs and previous studies have demonstrated that IL-2 induces selective expansion of CD4Tregs and improves clinical manifestations of chronic GVHD. However, mechanisms for selective activation of CD4Tregs and the effects of low-dose IL-2 on other immune cells are not well understood. Using mass cytometry, we demonstrate that low concentrations of IL-2 selectively induce STAT5 phosphorylation in Helios+ CD4Tregs and CD56brightCD16- NK cells in vitro. Preferential activation and expansion of Helios+ CD4Tregs and CD56brightCD16- NK cells was also demonstrated in patients with chronic GVHD receiving low-dose IL-2. With prolonged IL-2 treatment for 48 weeks, phenotypic changes were also observed in Helios- CD4Tregs. The effects of low-dose IL-2 therapy on conventional CD4+ T cells and CD8+ T cells were limited to increased expression of PD-1 on effector memory T cells. These studies reveal the selective effects of low-dose IL-2 therapy on Helios+ CD4Tregs and CD56bright NK cells that constitutively express high-affinity IL-2 receptors as well as the indirect effects of prolonged exposure to low concentrations of IL-2 in vivo.