Mouse Sertoli cells sustain de novo generation of regulatory T cells by triggering the notch pathway through soluble JAGGED1.

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.

Notch-Signaling Deregulation Induces Myeloid-Derived Suppressor Cells in T-Cell Acute Lymphoblastic Leukemia.

Notch receptors deeply influence T-cell development and differentiation, and their dysregulation represents a frequent causative event in "T-cell acute lymphoblastic leukemia" (T-ALL). "Myeloid-derived suppressor cells" (MDSCs) inhibit host immune responses in the tumor environment, favoring cancer progression, as reported in solid and hematologic tumors, with the notable exception of T-ALL. Here, we prove that Notch-signaling deregulation in immature T cells promotes CD11b+Gr-1+ MDSCs in the Notch3-transgenic murine model of T-ALL. Indeed, aberrant T cells from these mice can induce MDSCs in vitro, as well as in immunodeficient hosts. Conversely, anti-Gr1-mediated depletion of MDSCs in T-ALL-bearing mice reduces proliferation and expansion of malignant T cells. Interestingly, the coculture with Notch-dependent T-ALL cell lines, sustains the induction of human CD14+HLA-DRlow/neg MDSCs from healthy-donor PBMCs that are impaired upon exposure to gamma-secretase inhibitors. Notch-independent T-ALL cells do not induce MDSCs, suggesting that Notch-signaling activation is crucial for this process. Finally, in both murine and human models, IL-6 mediates MDSC induction, which is significantly reversed by treatment with neutralizing antibodies. Overall, our results unveil a novel role of Notch-deregulated T cells in modifying the T-ALL environment and represent a strong premise for the clinical assessment of MDSCs in T-ALL patients.

NF-κB1 Regulates Immune Environment and Outcome of Notch-Dependent T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric malignancy that arises from the transformation of immature T-cell progenitors and has no definitive cure. Notch signaling governs many steps of T cell development and its dysregulation represents the most common causative event in the pathogenesis of T-ALL. The activation of canonical NF-κB pathway has been described as a critical downstream mediator of Notch oncogenic functions, through the sustaining of tumor cell survival and growth. The potential role of Notch/NF-κB partnership is also emerging in the generation and function of regulatory T cells (Tregs) in the context of cancer. However, little is known about the effects of combined mutations of Notch and NF-κB in regulating immune-environment and progression of T-ALL. To shed light on the topics above we generated double-mutant mice, harboring conventional knock-out mutation of NF-κB1/p50 on the genetic background of a transgenic model of Notch-dependent T-ALL. The immunophenotyping of double-mutant mice demonstrates that NF-κB1 deletion inhibits the progression of T-ALL and strongly modifies immune-environment of the disease. Double-mutant mice display indeed a dramatic reduction of pre-leukemic CD4+CD8+ (DP) T cells and regulatory T cells (Tregs) and, concurrently, the rising of an aggressive myeloproliferative trait with a massive expansion of CD11b+Gr-1+ cells in the periphery, and an accumulation of the granulocyte/monocyte progenitors in the bone-marrow. Interestingly, double-mutant T cells are able to improve the growth of CD11b+Gr-1+ cells in vitro, and, more importantly, the in vivo depletion of T cells in double-mutant mice significantly reduces the expansion of myeloid compartment. Our results strongly suggest that the myeloproliferative trait observed in double-mutant mice may depend on non-cell-autonomous mechanism/s driven by T cells. Moreover, we demonstrate that the reduction of CD4+CD8+ (DP) T cells and Tregs in double-mutant mice relies on a significant enhancement of their apoptotic rate. In conclusion, double-mutant mice may represent a useful model to deepen the knowledge of the consequences on T-ALL immune-environment of modulating Notch/NF-κB relationships in tumor cells. More importantly, information derived from these studies may help in the refinement of multitarget therapies for the disease.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation.

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through ß-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4+CD8+ cells, by a "thymus-autonomous" mechanism.