RESUMEN
Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Separación Celular/métodos , Complejo Mayor de Histocompatibilidad/inmunología , Biotina/química , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Línea Celular , Células Clonales , Dextranos/química , Citometría de Flujo , Colorantes Fluorescentes/química , Antígeno HLA-A2/química , Antígeno HLA-DR1/química , Antígeno HLA-DR1/metabolismo , Hemaglutininas Virales/metabolismo , Humanos , Insulina/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica , Precursores de Proteínas/metabolismo , Estreptavidina/química , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Telomerasa/metabolismoRESUMEN
High-throughput T cell receptor sequencing on sequentially banked blood samples from healthy individuals has shown that high-frequency clonotypes can remain relatively stable for up to 18 years, with minimal inflation, deflation, or turnover. These populations included T cell expansions specific for Epstein-Barr virus. Thus, in spite of exposure to a barrage of microorganisms over the course of life, the dominant clonotypes in the mature peripheral T cell repertoire can alter surprisingly little.