We have generated transgenic mice (A10Tg) overexpressing A Disintegrin And Metalloproteinase (ADAM10) on common lymphoid and B-cell progenitors. This prevents B-cell development and induces myeloid derived suppressor cell (MDSC) accumulation in the absence of an established tumor. As in tumor bearing mice, A10Tg MDSCs consist of both granulocytic and monocytic phenotypes that profoundly inhibit polyclonal (anti-CD3/anti-CD28) and antigen-specific pmel1 T-cell proliferation, produce high levels of VEGF, and suppress L-selectin expression on cytotoxic T cells. Injection of B16 melanoma revealed that A10Tg mice are highly susceptible to metastasis and exhibit minimal response to melanoma-directed Adoptive Immunotherapy (AIT) compared to littermate controls. However, AIT combined with gemcitabine, a chemotherapeutic that preferentially decreases MDSCs, was found to inhibit B16 metastasis. Our preliminary data indicates that, despite what is known in the literature, the suppressive ability of MDSCs are not dependent on the tumor microenvironment. Therefore, we will utilize A10Tg mice to conduct comparative studies between A10Tg MDSCs and tumor-derived MDSCs for mechanisms of suppression. We will also use in vitro culture assays with pre-conditioned media to determine the response of tumor-free A10Tg MDSCs to common TDFs for their migration potential and phenotypic alterations. Finally, we will analyze the mechanisms of MDSC mediated T cell suppression with common inhibitors of MDSC activity. We will also explore the development of MDSCs. Mixed bone marrow chimeras between A10Tg and wild type hematopoietic stem cells (HSCs) show this phenomenon to be cell-intrinsic. Since Notch is a regulator of hematopoiesis and a substrate for ADAM10 cleavage, the observed phenotype may be a consequence of perturbed Notch signalling. Inhibition of Notch pathways in wild type HSCs results in MDSC accumulation, mimicking the phenotype of the A10Tg mice. Additionally, ex vivo blockade of ADAM10 in A10Tg HSCs rescues B-cell development and decreases myeloid cell counts. Therefore, both ADAM10 and Notch signalling regulate MDSC development. We will analyze the intensity and temporal stage of Notch signalling in haematopoiesis using OP9-DL1 with varying levels of the Notch lignad. ADAM10 and 3-secretase inhibitors will be used to determine the differential effects of S2 and S3 cleavage of Notch on MDSC accumulation. Our in vivo model will utilize mice that express the activated Notch intracellular domain (NICD) following cre- mediated recombination. These animals will be crossed with VavCre mice expressing cre recombinase under the vav promoter, restricting NICD expression to hematopoietic stem cells. We expect these mice to exhibit the same phenotype observed in the A10Tg mice. As an alternative, ADAM10flox/flox mice will be crossed with VavCre mice to determine the consequences of ADAM10 deletion in hematopoiesis. A10Tg mice provide a novel model for the study of MDSC generation and function in a tumor-free environment.
We propose to study the development and function of myeloid derived suppressor cells (MDSCs) in a mouse model in which the overexpression of A Disintegrin And Metalloproteinase 10 (ADAM10) resulted in abrogated B-cell development and an accumulation of MDSCs. A10Tg MDSCs are phenotypically and functionally parallel to tumor-derived MDSCs. Unlike existing mouse models, our A10Tg mice allow us to study MDSCs in the absence of tumor. This work will enable us to elucidate the mechanisms of MDSC development and role of MDSCs in anti-tumor immunity, thereby contributing to novel therapeutic strategies.
|Martin, Rebecca K; Saleem, Sheinei J; Folgosa, Lauren et al. (2014) Mast cell histamine promotes the immunoregulatory activity of myeloid-derived suppressor cells. J Leukoc Biol 96:151-9|