Thymocytes between the β-Selection and Positive Selection Checkpoints Are Nonresponsive to IL-7 as Assessed by STAT-5 Phosphorylation

Abstract

Interleukin-7 is widely accepted as a major homeostatic factor involved in T cell development. To assess the IL-7 responsiveness of thymocytes involved in selection processes, we used a new sensitive flow cytometry-based assay to detect intracellular phosphorylation of STAT-5 induced by IL-7 in defined mouse thymocyte subsets. Using this method, we found the earliest thymocyte subset (CD4−CD8−CD25−CD44+) to contain both IL-7-responsive and nonresponsive cells. Transition through the next stages of development (CD4−CD8−CD25+CD44+ and −) was associated with responsiveness of all thymocytes within these populations. Passage of thymocytes through β-selection resulted in a significant reduction in IL-7 sensitivity. In the next phases of development (TCR− and TCRlowCD69−), thymocytes were completely insensitive to the effects of IL-7. STAT-5 phosphorylation in response to IL-7 was again observed, however, in thymocytes involved in the positive selection process (TCRlowCD69+ and TCRintermediate). As expected, CD4 and CD8 single-positive thymocytes were responsive to IL-7. These findings delineate an IL-7-insensitive population between the β-selection and positive selection checkpoints encompassing thymocytes predicted to die by neglect due to failure of positive selection. This pattern of sensitivity suggests a two-signal mechanism by which survival of thymocytes at these checkpoints is governed.

Cell Surface Expression of the Bovine Leukemia Virus-Binding Receptor on B and T Lymphocytes Is Induced by Receptor Engagement

“…In a situation analogous to that of Glut1, we determined that the BLV-binding receptor is expressed on only a minority of freshly isolated human thymocytes (10 -20%), largely within the most immature DP thymocyte subset. IL-7, a cytokine that is crucial for thymocyte differentiation and proliferation, has previously been shown to induce responses in immature DN and mature SP cells, but not DP cells (61)(62)(63). In agreement with these data, we find that those IL-7-responsive human thymocytes, monitored as a function of their size and blast formation, are in vast majority SP and DN cells, excluding the DP population.…”

Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells

“…IL-7R␣ is expressed in developing B cells but is down-regulated upon B cell maturation (7,8). In developing T cells, IL-7R␣ is initially down-regulated at the CD4 and CD8 double negative 3 stage of thymic development (9). However, Il7r transcription is re-activated after the CD4 and CD8 double-positive stage and is expressed on peripheral CD4 or CD8 single-positive T cells (7).…”

Programmed Cell Death in T Cell Development

“…This is due to the fact that post-β-selection DN3 and DN4 thymocytes have decreased expression of IL-7Rα and subsequently reduced responsiveness to IL-7 compared to pre-β-selection stages (Van De Wiele et al 2004;Yu et al 2004;Van de Wiele et al 2007;Teague et al 2010). Interestingly, transgenic IL-7Rα expression ultimately impairs differentiation into DP thymocytes, suggesting that progression through β-selection is impeded by IL-7 signaling (Yu et al 2004).…”

“…DN2 and DN3) thymocytes, suggesting that IL-7 is needed for the transition to and/or at these stages (Peschon et al 1994;Maraskovsky et al 1997). Later studies that examined sorted DN populations confirm that the DN2 and DN3 subsets have the highest expression of IL-7Rα (Yu et al 2004) and responsiveness to IL-7 stimulation (Van De Wiele et al 2004). IL-7 promotes thymocyte survival through both positive regulation of anti-apoptotic members and negative regulation of pro-apoptotic members of the B cell lymphoma (Bcl) 2 family (for a summary of the role of Bcl-2 family members in T cell development, refer to Table 1).…”

“…TCR signaling during positive selection results in reacquisition of IL-7 responsiveness in post-selection DP and SP thymocytes (Van De Wiele et al 2004;Marino et al 2010). Neutralizing IL-7Rα has been shown to inhibit SP development upon transfer of thymocytes from a non-selection (MHC -/-) to a positive selection (MHC +/+ ) background (Akashi et al 1997).…”