Ten-color flow cytometry reveals distinct patterns of expression of CD124 and CD126 by developing thymocytes

We have developed a 12-parameter/10-color flow cytometric staining method for the simultaneous detection and characterization of 21 mouse thymocyte subpopulations that represent discreet stages of T cell development. To demonstrate the utility of this method, we assessed cytokine receptor expression on mouse thymocyte subsets. These experiments revealed distinct patterns of surface expression of receptors for the cytokines IL-4 and IL-6. The IL-4 receptor α chain (CD124) was highly expressed on the earliest thymocyte subsets, then downregulated prior to T cell receptor β-selection and finally upregulated in the CD4/CD8 double positive cells prior to positive selection. The IL-6 receptor α chain (CD126) showed a different pattern of expression. It was expressed on the most mature subsets within the CD4 and CD8 single positive (SP) compartments and was absent on all other thymocytes with the exception of a very small cKit-CD4-CD8- population. Intracellular staining of SP thymocytes for phosphorylated STAT-1 demonstrated that IL-6 signaling was confined to the most mature SP subsets. This 12-parameter staining methodology uses only commercially available fluorochrome-coupled monoclonal antibodies and therefore could be employed by any investigator with access to a 4-laser flow cytometer. This novel staining scheme allowed us to easily phenotype thymocyte subpopulations that span across development, from the early thymic progenitors (ETPs) to the most mature subsets of the CD4 and CD8 single positive populations.