Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33's immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif-bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

Allelic deletion of the RPS14 gene is a key effector of the hypoplastic anemia in patients with myelodysplastic syndrome (MDS) and chromosome 5q deletion [del(5q)]. Disruption of ribosome integrity liberates free ribosomal proteins to bind to and trigger degradation of MDM2, with consequent p53 transactivation. Herein we show that p53 is overexpressed in erythroid precursors of primary bone marrow del(5q) MDS specimens accompanied by reduced cellular MDM2. More importantly, we show that lenalidomide acts to stabilize MDM2, thereby accelerating p53 degradation. Biochemical and molecular analyses showed that lenalidomide inhibits the haplodeficient PP2Acα phosphatase resulting in hyperphosphorylation of inhibitory serine-166 and serine-186 residues on MDM2, and displaces binding of RPS-14 to suppress MDM2 auto-ubiquitination; whereas PP2Acα over expression promotes drug resistance. Bone marrow specimens from del(5q) MDS patients resistant to lenalidomide over-expressed PP2Acα accompanied by restored accumulation of p53 in erythroid precursors. Our findings indicate that lenalidomide restores MDM2 functionality in the 5q- syndrome to overcome p53 activation in response to nucleolar stress, and therefore may warrant investigation in other disorders of ribosomal biogenesis.

3, 5, 7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3- methylbutyl)–flavone (ICT) is a novel derivative of Icariin (ICA), the major active ingredient of Herba Epimedii, a herb used in traditional Chinese and alternative medicine. We previously demonstrated its anti-inflammatory effect in murine innate immune cells and activated human PBMCs. We report herein that ICA or ICT treatment reduces the expression of MRP8/MRP14 and toll-like receptor 4 (TLR4) on human PBMCs. Administration of ICA or ICT inhibited tumor growth in 4T1-Neu tumor-bearing mice and considerably decreased MDSC numbers in the spleen of these mice. Further, we saw a restoration of IFN-γ production by CD8+ T cells in tumor bearing mice when treated with ICA or ICT. ICA and ICT significantly decreased the amounts of nitric oxide and reactive oxygen species in MDSC in vivo. When MDSC were treated in vitro with ICT, we saw a significant reduction in the percent of these cells with concomitant differentiation into dendritic cells and macrophages. Concomitant with this cell type conversion was a down-regulation of IL-10, IL-6 and TNF-α production. Decreased expression of S100A8/9 and inhibition of activation of STAT3 and AKT may in part be responsible for the observed results. In conclusion, our results showed that ICA, and more robustly, ICT, directly modulate MDSC signaling and therefore altered the phenotype and function of these cells, in vitro and in vivo.

Previously, we showed that 2B4 is a dominant inhibitory receptor in SHIP-deficient NK cells that prevents efficient cytolysis of complex targets. We show in this study that 2B4 deficiency restores homeostatic control and cytolytic function to SHIP-deficient NK cells. However, 2B4−/−SHIP−/− NK cells still exhibit a profound disruption of their NK receptor repertoire and are compromised for induction of IFN-γ by several NK-activating receptors, including NKp46, NK.1.1, and NKG2D. In addition, we find that 2B4−/− NK cells have an extensively disrupted repertoire, including a supernormal frequency of NKp46+ NK cells. Consequently IFN-γ is induced on a much higher percentage of 2B4−/− NK cells following engagement of NKp46. We also find that both SHIP and 2B4 are required to prevent expression of Ly49B, a myeloid lineage MHC class I receptor not normally expressed by the NK lineage. Finally, when SHIP-deficient NK cells are on an H-2d background, they exhibit supernormal levels of Ly49A and possess normal cytolytic function against MHC-matched tumor targets and enhanced cytolysis of MHC mismatched tumor targets. However, despite normal or elevated cytolytic function, H2d SHIP−/− NK cells exhibit poor induction of IFN-γ like their H2b+ or 2B4−/− counterparts, demonstrating a uniform requirement for SHIP in induction of IFN-γ downstream of key NK activating receptors. These findings reveal a complex interplay of SHIP, 2B4, and MHC in the regulation of homeostasis, effector function, and repertoire formation in the NK cell lineage.