RESUMO
Highly multiplexed, single-cell technologies reveal important heterogeneity within cell populations. Recently, technologies to simultaneously measure expression of 96 (or more) genes from a single cell have been developed for immunologic monitoring. Here, we report a rigorous, optimized, quantitative methodology for using this technology. Specifically: we describe a unique primer/probe qualification method necessary for quantitative results; we show that primers do not compete in highly multiplexed amplifications; we define the limit of detection for this assay as a single mRNA transcript; and, we show that the technical reproducibility of the system is very high. We illustrate two disparate applications of the platform: a "bulk" approach that measures expression patterns from 100 cells at a time in high throughput to define gene signatures, and a single-cell approach to define the coordinate expression patterns of multiple genes and reveal unique subsets of cells.
Assuntos
Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Ativação Linfocitária/genética , Subpopulações de Linfócitos/imunologia , Reação em Cadeia da Polimerase Multiplex , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Animais , Primers do DNA , Regulação da Expressão Gênica , Marcadores Genéticos , Humanos , Limite de Detecção , Modelos Lineares , Macaca mulatta , Reprodutibilidade dos TestesRESUMO
Long-lived memory T cells are able to persist in the host in the absence of antigen; however, the mechanism by which they are maintained is not well understood. Recently, a subset of human T cells, stem cell memory T cells (TSCM cells), was shown to be self-renewing and multipotent, thereby providing a potential reservoir for T cell memory throughout life. However, their in vivo dynamics and homeostasis still remain to be defined due to the lack of suitable animal models. We identified T cells with a TSCM phenotype and stem cell-like properties in nonhuman primates. These cells were the least-differentiated memory subset, were functionally distinct from conventional memory cells, and served as precursors of central memory. Antigen-specific TSCM cells preferentially localized to LNs and were virtually absent from mucosal surfaces. They were generated in the acute phase of viral infection, preferentially survived in comparison with all other memory cells following elimination of antigen, and stably persisted for the long term. Thus, one mechanism for maintenance of long-term T cell memory derives from the unique homeostatic properties of TSCM cells. Vaccination strategies designed to elicit durable cellular immunity should target the generation of TSCM cells.