High levels of IL-7 cause dysregulation of thymocyte development

Abstract

IL-7 signaling is required for thymocyte development and its loss has a severe deleterious effect on thymus function. Thymocyte-stromal cell interactions and other mechanisms tightly regulate IL-7 expression. We show that disruption of that regulation by over-expression of IL-7 inhibits T-cell development and promotes extensive B-cell lymphopoiesis in the thymus. Our data reveal that high levels of IL-7 negate Notch-1 function in thymocytes found in IL-7 transgenic mice and in co-culture with OP9-DL1 cells. While high levels of IL-7R are present on thymocytes, increased suppressor of cytokine signaling-1 expression blunts IL-7 downstream signaling, resulting in hypo-phosphorylation of proteins in the PI3K-Akt pathway. Consequently, GSK3β remains active and inhibits Notch-1 signaling as observed by decreased Hes-1 and Deltex expression in thymic progenitors. This is the first demonstration that high levels of IL-7 antagonize Notch-1 signaling and suggest that IL-7 may affect T- versus B-lineage choice in the thymus.

Fig. 1.

Analysis of αβ thymocytes and B cells in the thymi of IL-7 Tg mice. (A) Absolute counts of pro-B (CD43⁺), pre-B and late pro-B (BP-1⁺), immature/mature B cells (IgM⁺) and total B220⁺ in the thymus of 4-week- (black bars) or 6- to 12-week- (grey bars) old IL-7 Tg mice and 6- to 12-week- (outlined bar) old WT mice are shown as mean ± standard error (10 mice in each group, *P < 0.0001). (B) IL-7R expression on thymocytes comprising the DN1 population (Lin⁻CD44⁺CD25⁻c-Kit⁺) in IL-7 Tg and WT mice (open histograms) compared with the isotype control (filled histograms), the mean fluorescence intensity (MFI) is shown below the histograms. The full gating for the FACS is shown in Supplemental Figure 1B, available at International Immunology Online. Data are representative of five experiments with three mice for each group. (C) Analysis of thymic B cells in mixed BM chimeras. The plots show thymic B220⁺ cells derived from CD45.1⁺ and CD45.1⁻ BM cells in both recipients. (D) Average numbers of thymic CD45.1⁺ B cells obtained when CD45.1⁺ WT BM were co-injected with IL-7 Tg or WT CD45.1⁻ cells. n = 7 mice in each group (*P value of 0.022). (E) Absolute number of thymic B cells in IL-7 Tg mice that received neutralizing anti-IL-7 mAb M25 (black bars) versus control CC57 (grey bars) are shown. (*P values of 0.03, 0.005 and 0.008 for total B220⁺, pro-B (CD43⁺) and pre-B (CD43⁻BP-1⁺) in the M25-treated IL-7 Tg mice, respectively). Each group included four mice. (F) ETPs in the thymus of 4-week-old IL-7 Tg and WT mice (*P = 0.0019; n = 9–13 mice). (G) Average numbers of B220⁺ or CD90⁺ cells obtained from co-cultures of ETPs sorted from either WT or IL-7 Tg mice and OP9-DL1 cells are shown from quadruplicate wells (P < 0.001).

Fig. 2.

Increase of cells with B-cell markers instead of DP cells on OP9-DL1 stromal cells in presence of high IL-7. (A) T- and B-cell development from WT or IL-7 Tg-LSKs co-cultured with OP9-DL1 cells. The percentages of DP, DN1-4 and B220/AA4.1 cells are shown in the corresponding quadrants. Representative of experiments repeated three times. (B) Numbers of B220⁻AA4.1⁺ (grey bars) and B220⁺AA4.1⁺ (black bars) obtained from IL-7 Tg-LSKs cultured on OP9-DL1 cells compared with WT-LSKs. (*P values for B220⁻AA4.1⁺ and B220⁺AA4.1⁺ are 0.0014 and 0.02 on OP9-DL1, respectively). The experiment was repeated three times. (C) Thymic-CLP-2 (T-CLP-2) in 2-week-old IL-7 Tg mice and WT littermates. The percentage of T-CLP-2 are shown, as well as the profiles of Flt-3 and Sca-1 markers on CLP-2 from IL-7 Tg thymus (open histograms) compared with isotype controls (shaded histograms). The results were confirmed in eight mice in each group included in three experiments. (D) Numbers of B220⁻AA4.1⁺ (grey bars) and B220⁺AA4.1⁺ (black bars) obtained from OP9-DL1 and LSK-Flt3⁺ WT progenitors co-cultures in proportion to the increasing doses of IL-7 (*P < 0.0001 in all groups). The experiment was repeated three times. (E) FACS analysis of B cells obtained from WT LSK-Flt3⁺ cells co-cultured with OP9 or OP9-DL1 cells and treated with different levels of IL-7. The open histograms show the profile of AA4.1⁺ cells on B220⁻CD19⁺ (left histogram) and B220⁺CD19⁻ cells (right histogram) compared with the isotype control (shaded histograms) from the sample treated with 40ng ml⁻¹ IL-7. The numbers below the histograms represent the MFI. The experiment was repeated twice.

Fig. 3.

Gene expression analysis of cells exposed to high IL-7 doses. (A) Panels compare the relative gene expression of IL-7 Tg and WT. Left panel, LRF expression in LSK-Flt3⁺ cells; middle panel, Bcl11b and TCF-7 expression in DN1 c-Kit⁺ cells; right panel, EBF and Pax5 expression in DN1 c-Kit⁺ cells (*P < 0.001). (B) Left panel, relative expression levels of Hes-1 and Deltex expression in DN1 c-Kit⁺ cells from WT and IL-7 Tg mice (*P < 0.0001 for Hes-1 and 0.0008 for Deltex). Right panel, relative expression levels of Hes-1 and Deltex expression in DN1 c-Kit⁺ cells sorted from WT LSK-Flt3⁺ and cultured with OP9-DL1 cells at 1, 10 or 40ng ml⁻¹ of IL-7 (Hes-1 *P < 0.05, Deltex *P < 0.0001). Data shown in (A) n = 8–9 mice each group done in triplicate and (B) are from two experiments done in triplicate. (C) LSK-Flt-3⁺ progenitors were sorted from IL-7 Tg and WT mice. WT cells were cultured on OP9-DL1 stromal cells with 1ng ml⁻¹ (WT-1) or 40ng ml⁻¹ of IL-7 (WT-40). IL-7 Tg cells were cultured on OP9-DL1 stromal cells with 1ng ml⁻¹ IL-7 (IL-7 Tg). After culture, DN1 c-Kit⁺, DN2 c-Kit⁺ and DN3 c-Kit⁻ progenitors were separately sorted then mixed in the same proportions for each group. Total and phospho-Akt Ser⁴⁷³ are shown for all groups. Phospho-Akt-Thr³⁰⁸ is shown in IL-7 Tg and WT-derived cells. (D) Normalized SOCS-1 expression in sorted DN1-c-Kit⁺, DN2-ckit⁺ and DN3-c-Kit⁻ from WT LSK-Flt-3⁺ and OP9-DL1 cell co-cultures treated with1ng ml⁻¹ or 40ng ml⁻¹ of IL-7. IL-7 Tg LSK-Flt3⁺ were cultured with 1ng ml⁻¹ of IL-7 (*P < 0.0001). (E) Normalized SOCS-1 expression in sorted DN3-c-Kit⁻ and DN4-c-Kit⁻ thymocytes from IL-7 Tg and WT mice (*P = 0.0004; **P = 0.0001). (F) STAT5 intracellular staining in DN thymocytes from IL-7 Tg and WT mice. The black filled histograms show the isotype control for STAT5 staining; the grey histograms show STAT5 staining for unstimulated cells; the open histograms correspond to STAT5 staining in progenitors stimulated with IL-7. Gates were DN1-c-Kit⁺, DN2-ckit⁺, DN3-c-Kit⁻ and DN4-c-Kit⁻ within the Lin⁻ population. The experiment was repeated three times.

Fig. 4.

PI3K-Akt pathway controls Notch signaling through GSK3β. (A) Western blots for total and phospho-GSK3β, PTEN and Akt were performed on normalized populations of DN1 c-Kit⁺, DN2 c-Kit⁺, and DN3 c-Kit⁻ progenitors freshly sorted from WT and IL-7 Tg mice. (B) Total counts of DN1 c-Kit⁺ (grey bars) and DN3 c-Kit⁻ (black bars) cells after culture of LSKs in presence of normal or high IL-7 doses with or without lithium chloride. LSKs from WT mice were treated with 1, 10 or 40ng ml⁻¹ while LSK from IL-7 Tg mice were cultured with 1ng ml⁻¹ IL-7. Cell numbers from all cultures are compared with WT cultures with 1ng ml⁻¹ of IL-7. The results represent three combined experiments (*P = 0.02). (C) DN1-4 profiles after culture of IL-7 Tg or WT progenitors with or without LiCl and different IL-7 doses. The percentages of all DN stages are shown from one out of three representative experiments.