Differential IL-7 responses in developing human thymocytes

Abstract

Interleukin (IL)-7 is a factor essential for mouse and human thymopoiesis. Mouse thymocytes have altered sensitivities to IL-7 at different developmental stages. CD4/CD8 double positive (DP) mouse thymocytes are shielded from the influence of IL-7 because of loss of CD127 (IL-7Ralpha). In this study, we assessed IL-7 receptor expression and IL-7 signaling in human thymocytes. We found human DP cells to be severely limited in their ability to phosphorylate STAT-5 in response to IL-7. The relative expression levels of the IL-7-inducible proteins Bcl-2 and Mcl-1 were also lower in human DP cells, consistent with a stage-specific decrease in IL-7 responsiveness. IL-7 responses were restored in a subset of cells that matured past the DP stage. Unlike the regulation of IL-7 signaling in mouse thymocytes, loss of IL-7 signaling in human DP cells was not due to absence of CD127, but instead correlated with downregulation of CD132 (common gamma chain).

Fig. 1. IL-7Rα and common γ receptor expression and IL-7 signaling in the TN, ISP4, DP^CD3lo DP^CD3hi, SP4 and SP8 thymocyte subsets

IL-7Rα and common γ receptor expression levels were evaluated on gated thymocytes by staining with antibodies to CD3, CD4, CD8, CD127, and CD132 (left panels). Open histograms represent staining with isotype control mAb and closed histograms represent IL-7Rα (CD127) and common γ receptor (CD132) staining. Equivalent cell numbers from each subset were treated with (open histograms) or without (closed histograms) IL-7 and then analyzed for intracellular levels of pSTAT-5 (right panels). The data in this figure are from patient #1, detailed in Table 1.

Fig. 2. Western blotting confirms that DP thymocytes do not respond to IL-7 via the STAT-5 signaling pathway

Lysates of equivalent cell numbers from sorted subsets (TN, DP^CD3lo, DP^CD3hi, SP4 and SP8) were treated with IL-7, IFN-γ, or medium alone and then analyzed by Western blotting for pSTAT-5, or β-actin. The loss of pSTAT-5 response in the DP cells was seen in samples from all 3 patients for which this assay was performed.

Fig. 3. Mcl-1 and Bcl-2 protein expression is diminished in human DP thymocytes

Human thymocytes were sorted into six major subpopulations (TN, ISP4, DP^CD3lo, DP^CD3hi, SP4, and SP8). Sorted cells (500,000 cells/lane) were lysed and blotted with antibodies against Mcl-1, Bcl-2, and β-actin as described in the methods sections. Shown are three separate experiments (A, B, and C) from 3 individual patients.