Within the thymic microenvironment, integration of Notch-1 and the IL-7 signaling pathways are crucial to support T-cell development. IL-7 expression in thymic epithelial cells is regulated by location, ageing and thymocyte interactions (4–6,29,30). Given the importance of IL-7 and Notch signaling on thymus function, it seemed likely that efficient thymocyte development might depend on cross-talk between these pathways. Therefore, alterations in bioavailability of IL-7, e.g. during high dose clinical treatments, may affect T-cell development. Support for this concept was provided by a study showing that IL-7 enhances proliferation of early thymocytes while opposing Notch-1-dependent progression beyond the DN3 stage in OP9-DL1 co-cultures (31
). Another example was the finding that Notch-1 signaling potentiates IL-7 signaling in human thymocytes grown on OP9-DL1 (32
). Together, data from these in vitro
studies suggest that regulatory circuits do exist between the IL-7 and Notch pathways.
To investigate the effect of clinically therapeutic levels of IL-7 in vivo
, we created a Tg mouse that expresses about 40-fold higher than normal levels of IL-7 in the thymus (17
). Unexpectedly, high IL-7 levels reduced αβ T-cell development (17
), while promoting thymic B-cell development. Our findings differ from data using a different IL-7 Tg where B-lineage cells entered the thymus from the circulation due to very high levels of B lymphopoiesis in spleen, lymph nodes, and blood (33
). In contrast, we propose that high levels of IL-7 inhibited Notch based on our observation of numerous phenotypic abnormalities observed in the IL-7 Tg thymus and known to be associated with decreased Notch-1 signaling: fewer T cells and more B cells in the thymus; (7
) reduced numbers of ETPs, DN2 and pre T cells and increases in γδ T cells and NK cells (18
). This idea was strengthened when we observed decreased steady state levels of the Notch-1 target genes Hes-1 and Deltex in ETP cells. Together, our data strongly suggested that a major effect of high IL-7 on thymocytes is an active inhibition of Notch-1 signaling in spite of abundant DLL1 or DLL4 in the thymus and in our OP9-DL1 co-cultures.
We next examined signal transduction pathways to determine how Notch signaling was reduced and determined that PI3K, Akt and GSK3β are hypo-phosphorylated in IL-7 Tg thymocytes compared with controls. These data provided insight toward a potential mechanism because it is known that GSK3β phosphorylates and inhibits Notch (10
). Based on this idea, we showed that LiCl inactivation of GSK3β (10
) in cells exposed to high IL-7 levels resulted in restoration of normal thymocyte development in OP9-DL co-cultures. Together, our data strongly suggested that a major effect of high IL-7 on thymocytes is an active inhibition of Notch-1 signaling.
Our results indicate that high levels of IL-7 also skew signaling networks and alter the developmental potential of thymocyte precursor cells. For example, we observed an increase in SOCS-1 expression in IL-7 Tg c-Kit+
DN1 cells. In accord with SOCS-1 ability to blunt cytokine responses (35
), we also found that phospho-STAT5 levels were reduced in IL-7 Tg DN1-4 cells compared with WT controls. This finding distinguishes our work from others that showed that expression of constitutively active STAT5 drives thymic B-cell development (36
In addition, we documented an increase in EBF and Pax5, master regulators of B-cell development in DN1 c-Kit+
cells sorted from IL-7 Tg mice. Conversely, we showed a dramatic decrease in Bcl11b and TCF-7, two genes critical for T-cell lineage commitment and development in DN1 c-Kit+
cells sorted from IL-7 Tg mice. Of particular interest is the recent finding that Bcl11b expression is repressed in culture conditions with high levels of IL-7 and Notch ligand (25
). In these conditions, DN2 cells that retain myeloid and T-cell (but not B-cell) potential accumulate but are unable to undergo T-cell development until IL-7 levels are reduced (25
). Although our data suggest that cells from an earlier developmental stage are diverted to thymic B-cell development in the IL-7 Tg, it seems likely that repression of Bcl11b by high IL-7 contributes to the inefficiency of T-cell production observed in vivo
and in vitro
. Together, these data are consistent with a diversion from T- to B-lineage development of IL-7 Tg thymic precursors.
One intriguing question is the nature of the cells that give rise to the abnormal thymic B-cell development in our Tg model. The majority of the earliest thymic precursor cells has a history of IL-7R expression (2
) that may affect lineage choice in the thymus. Using the OP9-DL1 co-culture system, we tested different progenitors in the IL-7 Tg for B-cell potential. ETPs did not appear to be the source of the thymic B cells observed in the IL-7 Tg mice. However, we identified CLP-2 cells in the thymus of IL-7 Tg mice that was absent in WT mice, possibly representing a rare population of independent progenitor cells seeding the thymus (20
). We also observed a distinctive population of B220+
cells that developed from LSK progenitors in the presence of high IL-7 and Notch-1 ligand (37
). These cells are similar to the B220+
cells described in vivo
as pro-T cells that convert to B cells upon Notch-1 deletion (38
). CLP-2 cells normally die in the thymus in response to Notch signaling (39
). Together, these findings suggest that the abnormal B-cell development in the IL-7 Tg thymus results from inhibition of Notch-1 signaling in lymphoid progenitors. This idea was supported by the reduced number and T-cell potential of IL-7 Tg ETP cells in spite of the presence of Notch ligand. In addition, TCRαβ differentiation in IL-7 Tg mice is blocked at the DN3 stage, a point where it has been reported that high Notch levels are required for survival and expansion (40
). In addition, a down-regulation of Notch-1 signal may lead to accumulation of CLP-2 progenitors in the IL-7 Tg thymus and may constitute an alternative progenitor population giving rise to the B cells and thymocytes found in the IL-7 Tg thymus. The increase of LRF expression in LSK Flt3+
BM cells in IL-7 Tg mice could also explain the variability of thymic seeding progenitors in their T versus B potential leading to less T and more B cells in the thymus.
These findings demonstrate for the first time that high levels of IL-7 antagonize Notch signaling and dysregulate thymus function. It is likely that this information will lead to new understandings regarding lineage choice and expansion of precursor cells. For example, precursor cells are exposed to differing levels of IL-7 due to gradients formed by clusters of cells expressing higher levels of IL-7 in the thymus and other anatomical sites (5
). Because our work suggests that IL-7 mediates an environmental regulation of Notch signaling, differences in localized IL-7 availability in niches such as BM and thymus may be an important component of B–T lineage development. It may also explain the functional importance of thymocyte down-regulation of IL-7 expression in thymic stromal cells (6
). Our findings also present new considerations for patient treatment since IL-7 has been introduced in clinical settings (15
). Optimal clinical effects on peripheral expansion occurred at levels 1000-fold over baseline (15
), which corresponds to the high levels of IL-7 where we observed negative effects on thymic function in our experimental models. Based on our data, we hypothesize that high IL-7 levels would diminish thymus function and may preclude simultaneous augmentation of T-cell populations by thymic dependent and peripheral expansion pathways by high dose IL-7 treatment. This may be an especially important consideration for pediatric patients because they often exhibit robust thymic function following hematopoietic stem cell transplantation