TGF-ß-inducible microRNA-183 silences tumor-associated natural killer cells.

Transforming growth factor ß1 (TGF-ß), enriched in the tumor microenvironment and broadly immunosuppressive, inhibits natural killer (NK) cell function by yet-unknown mechanisms. Here we show that TGF-ß-treated human NK cells exhibit reduced tumor cytolysis and abrogated perforin polarization to the immune synapse. This result was accompanied by loss of surface expression of activating killer Ig-like receptor 2DS4 and NKp44, despite intact cytoplasmic stores of these receptors. Instead, TGF-ß depleted DNAX activating protein 12 kDa (DAP12), which is critical for surface NK receptor stabilization and downstream signal transduction. Mechanistic analysis revealed that TGF-ß induced microRNA (miR)-183 to repress DAP12 transcription/translation. This pathway was confirmed with luciferase reporter constructs bearing the DAP12 3' untranslated region as well as in human NK cells by use of sense and antisense miR-183. Moreover, we documented reduced DAP12 expression in tumor-associated NK cells in lung cancer patients, illustrating this pathway to be consistently perturbed in the human tumor microenvironment.

Tipifarnib-mediated suppression of T-bet-dependent signaling pathways.

Large granular lymphocyte (LGL) leukemia is a chronic lymphoproliferative disease in which T-bet [T-box transcription factor 21 gene (tbx21)] overexpression may play a pathogenic role. T-bet orchestrates the differentiation of mature peripheral T-cells into interferon-γ (IFN-γ) and tumor necrosis factor-α producing CD4+ T-helper type I (Th1) and CD8+ T cytotoxic cells that are necessary for antiviral responses. When IL-12 is produced by antigen-presenting cells, T-bet expression is induced, causing direct stimulation of ifng gene transcription while simultaneously acting as a transcriptional repressor of the IL4 gene, which then leads to Th1 dominance and T-helper type 2 differentiation blockade. Additionally, T-bet has been shown to regulate histone acetylation of the ifng promoter and enhancer to loosen condensed DNA, creating greater accessibility for other transcription factor binding, which further amplifies IFNγ production. We found that treatment with a farnesyltransferase inhibitor tipifarnib reduced Th1 cytokines in LGL leukemia patient T-cells and blocked T-bet protein expression and IL-12 responsiveness in T-cells from healthy donors. The mechanism of suppression was based on modulation of histone acetylation of the ifng gene, which culminated in Th1 blockade.

A critical role for phosphatase haplodeficiency in the selective suppression of deletion 5q MDS by lenalidomide.

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype, the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases, Cdc25C and PP2Acalpha, which are coregulators of the G(2)-M checkpoint, are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis, whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS.

A critical role for DAP10 and DAP12 in CD8+ T cell-mediated tissue damage in large granular lymphocyte leukemia.

Large granular lymphocyte (LGL) leukemia, or LGLL, is characterized by increased numbers of circulating clonal LGL cells in association with neutropenia, anemia, rheumatoid arthritis, and pulmonary artery hypertension (PAH). Emerging evidence suggests that LGLL cells with a CD8(+)CD28(null) phenotype induce these clinical manifestations through direct destruction of normal tissue. Compared with CD8(+)CD28(null) T cells from healthy controls, CD8(+)CD28(null) T cells from LGLL patients have acquired the ability to directly lyse pulmonary artery endothelial cells and human synovial cells. Here, we show that LGLL cells from patients possess enhanced cytotoxic characteristics and express elevated levels of activating natural killer receptors as well as their signaling partners, DAP10 and DAP12. Moreover, downstream targets of DAP10 and DAP12 are constitutively activated in LGLL cells, and expression of dominant-negative DAP10 and DAP12 dramatically reduces their lytic capacity. These are the first results to show that activating NKR-ligand interactions play a critical role in initiating the DAP10 and DAP12 signaling events that lead to enhanced lytic potential of LGLL cells. Results shown suggest that inhibitors of DAP10 and DAP12 or other proteins involved in this signaling pathway will be attractive therapeutic targets for the treatment of LGLL and other autoimmune diseases and syndromes.

Clinical improvement by farnesyltransferase inhibition in NK large granular lymphocyte leukemia associated with imbalanced NK receptor signaling.

Large granular lymphocyte (LGL) leukemia is commonly associated with poor hematopoiesis. The first case of pulmonary artery hypertension (PAH) was observed in a 57-year-old woman with natural killer (NK)-LGL leukemia and transfusion-dependent anemia. Using a genetic approach, we demonstrated that killing of pulmonary endothelial cells by patient NK cells was mediated by dysregulated balance in activating and inhibitory NK-receptor signaling. Elevated pulmonary artery pressure and erythroid differentiation improved after disrupting the NK-receptor signaling pathway with 4 courses of a farnesyltransferase inhibitor, tipifarnib. Coincidental association between PAH and LGL leukemia suggest a causal relationship between the expanded lymphocyte population and these clinical manifestations. This trial is registered at www.ClinicalTrials.gov as NCI 6823.

In contrast to anti-tumor activity, YT cell and primary NK cell cytotoxicity for Cryptococcus neoformans bypasses LFA-1.

NK cell cytotoxicity requires two positive signals for killing of tumors. Activation receptors induce polarization of the microtubule organization center and degranulation, while leukocyte function-associated antigen (LFA)-1 is required for conjugate formation and actin polymerization and under some circumstances may be sufficient for NK cell cytotoxicity. Although the receptor for direct killing of fungi is not known, CD18, the beta2 chain of LFA-1, binds components of the capsule and cell wall of the opportunistic pathogen Cryptococcus neoformans, namely the polysaccharides glucoronoxylomannan and galactoxylomannan. Herein, we also demonstrate that LFA-1 was concentrated in regions of the NK cell surface interacting with C. neoformans. Consequently, there was compelling evidence to hypothesize that NK cells would also use LFA-1 to recognize and kill C. neoformans. Using a combination of NK cell lines that did or did not express LFA-1 or by using a CD18-specific functional blocking antibody, we confirm that NK cell anti-tumor activity is critically dependent upon the expression of LFA-1. Duplicating the events of tumor cytotoxicity, NK cells form conjugates with cryptococcal targets, rearrange the cell cytoskeleton to develop an NK immunologic synapse and release perforin-containing granules; however, each of these events occurred independently of LFA-1. Furthermore, NK cell-mediated killing of C. neoformans was detectable in both NK cells pre-treated with CD18-blocking antibodies and in NK cells lacking cell surface LFA-1 expression. These results demonstrate that in the absence of LFA-1 expression, NK cells are fully capable of recognizing a target (C. neoformans) and retain all of the events required for cytotoxicity.

MicroRNA-155 governs SHIP-1 expression and localization in NK cells and regulates subsequent infiltration into murine AT3 mammary carcinoma.

NK cell migration and activation are crucial elements of tumor immune surveillance. In mammary carcinomas, the number and function of NK cells is diminished, despite being positively associated with clinical outcome. MicroRNA-155 (miR-155) has been shown to be an important regulator of NK cell activation through its interaction with SHIP-1 downstream of inhibitory NK receptor signaling, but has not been explored in regard to NK cell migration. Here, we explored the migratory potential and function of NK cells in subcutaneous AT3 in mice lacking miR-155. Without tumor, these bic/miR-155-/- mice possess similar numbers of NK cells that exhibit comparable surface levels of cytotoxic receptors as NK cells from wild-type (WT) mice. Isolated miR-155-/- NK cells also exhibit equivalent cytotoxicity towards tumor targets in vitro compared to isolated WT control NK cells, despite overexpression of known miR-155 gene targets. NK cells isolated from miR-155-/- mice exhibit impaired F-actin polymerization and migratory capacity in Boyden-chamber assays in response chemokine (C-C motif) ligand 2 (CCL2). This migratory capacity could be normalized in the presence of SHIP-1 inhibitors. Of note, miR-155-/- mice challenged with mammary carcinomas exhibited heightened tumor burden which correlated with a lower number of tumor-infiltrating NK1.1+ cells. Our results support a novel, physiological role for SHIP-1 in the control of NK cell tumor trafficking, and implicate miR-155 in the regulation of NK cell chemotaxis, in the context of mammary carcinoma. This may implicate dysfunctional NK cells in the lack of tumor clearance in mice.

Immune evasion by TGFß-induced miR-183 repression of MICA/B expression in human lung tumor cells.

Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3'untranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3'UTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFß), as evidenced by antisense TGFß transfection into H1355 or H1299 tumor cells which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFß expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.

Bax-interacting factor-1 expression in prostate cancer.

BACKGROUND: Bax-interacting factor (Bif)-1 protein is a member of the endophilin B family that binds to and activates the proapoptotic Bax protein in response to apoptotic signals. Loss of Bif-1 suppresses the intrinsic pathway of apoptosis and promotes tumorigenesis. We examined the expression levels of Bif-1 protein in human prostate cancer. MATERIALS AND METHODS: Thirty-nine archival tissue specimens of human prostate cancer, and a human prostate cancer tissue microarray containing 19 samples of normal prostate, 26 samples of benign prostatic hyperplasias (BPHs), 30 samples of high-grade prostatic intraepithelial neoplasia (PIN), and 153 samples of prostate cancer, were selected for immunohistochemical staining with Bif-1 antibody. The slides were scored by 2 independent observers. RESULTS: Nontissue microarray samples: moderate to strong Bif-1 staining was identified in 38 of 39 prostate cancer samples. In 32 cases, foci of PIN were identified adjacent to prostate cancer samples. Of these, 29 samples (90.6%) showed strong and diffuse Bif-1 staining. Benign prostatic hyperplasias, identified in 27 cases, was weakly Bif-1 positive in 88.9% of cases. Tissue microarray samples: 38.6% (59 of 153) of prostate cancer samples showed moderate to strong Bif-1 expression, and 21.6% (33 of 153) were Bif-1 negative. Bif-1 expression was moderate to strong in 76.7% (23 of 30) of PIN. Bif-1 was weak to moderate in 53.8% (14 of 26) of BPH and negative in 46.2% (12 of 26) of them. Low to moderate Bif-1 was seen in 89.5% of normal prostate samples. CONCLUSION: The loss of Bif-1 expression in a subset of prostate cancer samples is in agreement with the proapoptotic function of Bif-1. The significance of the increased Bif-1 in a subgroup of prostate cancer samples and in PIN remains to be determined. It seems that Bif-1 has a role in prostate cancer, providing the rationale for using Bif-1 as a target for prostate anticancer therapy.

Clusterin mediates TRAIL resistance in prostate tumor cells.

One of the major obstacles in curing prostate cancer is the development of drug resistance to docetaxel, which is the gold standard for the treatment of this disease. It is not only imperative to discover the molecular basis of resistance but also to find therapeutic agents that can disrupt the resistant pathways. Based on initial findings that docetaxel-resistant PC3-DR and DU145-DR prostate tumor cell lines express tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, we examined whether TRAIL could be used as an alternative method to kill PC3-DR and DU145-DR cells. However, these tumor cells were found to be TRAIL resistant. Because PC3-DR and DU-145-DR cells were previously shown by us to be clusterin positive, we examined if clusterin could play a role in TRAIL resistance. We found that resveratrol could sensitize docetaxel-resistant tumor cells to TRAIL, and it worked by blocking clusterin expression. In particular, small interfering RNA clusterin expression in the cell lines was sufficient to produce apoptosis by TRAIL. Further analysis indicated that resveratrol functions as an effective tyrosine kinase inhibitor, similar to its analogue, piceatannol, and could inhibit Src and Jak kinases, thus resulting in loss of Stat1 activation. We have shown earlier that Stat1 is essential for gene transcription of clusterin. These results, taken together, show that resveratrol could be a useful new therapeutic agent to combat docetaxel resistance.

Inhibition of Human Dendritic Cell ER Stress Response Reduces T Cell Alloreactivity Yet Spares Donor Anti-tumor Immunity.

Acute graft- vs. -host disease (GVHD) is an important cause of morbidity and death after allogeneic hematopoietic cell transplantation (HCT). We identify a new approach to prevent GVHD that impairs monocyte-derived dendritic cell (moDC) alloactivation of T cells, yet preserves graft- vs.-leukemia (GVL). Exceeding endoplasmic reticulum (ER) capacity results in a spliced form of X-box binding protein-1 (XBP-1s). XBP-1s mediates ER stress and inflammatory responses. We demonstrate that siRNA targeting XBP-1 in moDCs abrogates their stimulation of allogeneic T cells. B-I09, an inositol-requiring enzyme-1α (IRE1α) inhibitor that prevents XBP-1 splicing, reduces human moDC migration, allo-stimulatory potency, and curtails moDC IL-1ß, TGFß, and p40 cytokines, suppressing Th1 and Th17 cell priming. B-I09-treated moDCs reduce responder T cell activation via calcium flux without interfering with regulatory T cell (Treg) function or GVL effects by cytotoxic T lymphocytes (CTL) and NK cells. In a human T cell mediated xenogeneic GVHD model, B-I09 inhibition of XBP-1s reduced target-organ damage and pathogenic Th1 and Th17 cells without impacting donor Tregs or anti-tumor CTL. DC XBP-1s inhibition provides an innovative strategy to prevent GVHD and retain GVL.

Akt mediates Ras downregulation of RhoB, a suppressor of transformation, invasion, and metastasis.

Although recent evidence supports a tumor-suppressive role for the GTPase RhoB, little is known about its regulation by signal transduction pathways. Here we demonstrate that Ras downregulates RhoB expression by a phosphatidylinositol 3-kinase (PI3K)- and Akt- but not Mek-dependent mechanism. Furthermore, genetic and pharmacological blockade of PI3K/Akt results in upregulation of RhoB expression. We also provide evidence for the importance of the downregulation of RhoB in oncogenesis by demonstrating that RhoB antagonizes Ras/PI3K/Akt malignancy. Ectopic expression of RhoB, but not the close relative RhoA, inhibits Ras, PI3K, and Akt induction of transformation, migration, and invasion and induces apoptosis and anoikis. Finally, RhoB inhibits melanoma metastasis to the lung in a mouse model. These studies identify suppression of RhoB as a mechanism by which the Ras/PI3K/Akt pathway induces tumor survival, transformation, invasion, and metastasis.

Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

Myeloid-derived suppressor cells (MDSCs) constitute a key checkpoint that impedes tumor immunity against cancer. Chemotherapeutic intervention of MDSCs has gained ground as a strategy for cancer therapy but its mechanism remains obscure.We report here a unique mechanism by which monocytic (M)-MDSCs are spared, allowing them to polarize towards M1 macrophages for reactivation of immunity against breast cancer. We first demonstrated that curcumin, like docetaxel (DTX), can selectively target CD11b(+)Ly6G(+)Ly6C(low) granulocytic (G)-MDSCs, sparing CD11b(+)Ly6G(-)Ly6C(high) M-MDSCs, with reduced tumor burden in 4T1-Neu tumor-bearing mice. Curcumin treatment polarized surviving M-MDSCs toward CCR7(+) Dectin-1(-)M1 cells, accompanied by IFN-γ production and cytolytic function in T cells. Selective M-MDSC chemoresistence to curcumin and DTX was mediated by secretory/cytoplasmic clusterin (sCLU). sCLU functions by trapping Bax from mitochondrial translocation, preventing the apoptotic cascade. Importantly, sCLU was only found in M-MDSCs but not in G-MDSCs. Knockdown of sCLU in M-MDSCs and RAW264.7 macrophages was found to reverse their natural chemoresistance. Clinically, breast cancer patients possess sCLU expression only in mature CD68(+) macrophages but not in immature CD33(+) immunosuppressive myeloid cells infiltrating the tumors. We thus made the seminal discovery that sCLU expression in M-MDSCs accounts for positive immunomodulation by chemotherapeutic agents.

Abscopal Benefits of Localized Radiotherapy Depend on Activated T-cell Trafficking and Distribution between Metastatic Lesions.

It remains unclear how localized radiotherapy for cancer metastases can occasionally elicit a systemic antitumor effect, known as the abscopal effect, but historically, it has been speculated to reflect the generation of a host immunotherapeutic response. The ability to purposefully and reliably induce abscopal effects in metastatic tumors could meet many unmet clinical needs. Here, we describe a mathematical model that incorporates physiologic information about T-cell trafficking to estimate the distribution of focal therapy-activated T cells between metastatic lesions. We integrated a dynamic model of tumor-immune interactions with systemic T-cell trafficking patterns to simulate the development of metastases. In virtual case studies, we found that the dissemination of activated T cells among multiple metastatic sites is complex and not intuitively predictable. Furthermore, we show that not all metastatic sites participate in systemic immune surveillance equally, and therefore the success in triggering the abscopal effect depends, at least in part, on which metastatic site is selected for localized therapy. Moreover, simulations revealed that seeding new metastatic sites may accelerate the growth of the primary tumor, because T-cell responses are partially diverted to the developing metastases, but the removal of the primary tumor can also favor the rapid growth of preexisting metastatic lesions. Collectively, our work provides the framework to prospectively identify anatomically defined focal therapy targets that are most likely to trigger an immune-mediated abscopal response and therefore may inform personalized treatment strategies in patients with metastatic disease.

ERK couples chronic survival of NK cells to constitutively activated Ras in lymphoproliferative disease of granular lymphocytes (LDGL).

Chronic NK lymphoproliferative disease of large granular lymphocytes (LDGL) is characterized by the expansion of activated CD3-, CD16+ or CD56+ lymphocytes. The mechanism of survival of NK cells from LDGL patients is unknown but may be related to antigenic stimulation. There is currently no standard effective therapy for LDGL, and the disease is characteristically resistant to standard forms of chemotherapy. We found evidence of constitutive activation of extracellular-regulated kinase (ERK) in NK cells from 13/13 patients with NK-LDGL (one patient with aggressive and 12 patients with chronic disease). Ablation of ERK activity by inhibitors or a dominant-negative form of MEK, the upstream activator of ERK, reduced the survival of patient NK cells. Ras was also constitutively active in patient NK cells, and exposure of cells to the Ras inhibitor FTI2153 or to dominant-negative-Ras resulted not only in ERK inhibition but also in enhanced apoptosis in both the presence and absence of anti-Fas. Therefore, we conclude that a constitutively active Ras/MEK/ERK pathway contributes to the accumulation of NK cells in patients with NK-LDGL. These findings suggest that strategies to inhibit this signaling pathway may be useful for the treatment of the NK type of LDGL.

A view to a kill: signals triggering cytotoxicity.

Tumor immunity requires the participation of lymphocyte effector cells that display powerful processes to destroy malignant cells. Natural killer (NK) cells and CTLs, once activated, use the same lytic processes for mediating target cell death. However, they are triggered through distinctly separate antigen receptors. NK cells are currently known to express at least three families of receptors unrelated to the T cell receptor, i.e., NKG2, KIR, and NCR, to mediate cytotoxicity. This review provides a view to a kill, bringing together a unifying concept for a common signal pathway that dictates lytic function. What emerges is a specific Syk/Zap70 --> PI3K --> Rac --> PAK --> MEK --> ERK signal cascade triggered by target cell recognition, which is responsible for mobilizing the lytic granules containing perforin and granzyme B toward the contacted target cell.

HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells.

Development of chemoresistance, especially to docetaxel (DTX), is the primary barrier to the cure of castration-resistant prostate cancer but its mechanism is obscure. Here, we report a seminal crosstalk between dying and residual live tumor cells during treatment with DTX that can result in outgrowth of a chemoresistant population. Survival was due to the induction of secretory/cytoplasmic clusterin (sCLU), which is a potent anti-apoptotic protein known to bind and sequester Bax from mitochondria, to prevent caspase 3 activation. sCLU induction in live cells depended on HMGB1 release from dying cells. Supernatants from DTX-treated DU145 tumor cells, which were shown to contain HMGB1, effectively induced sCLU from newly-plated DU145 tumor cells and protected them from DTX toxicity. Addition of anti-HMBG1 to the supernatant or pretreatment of newly-plated DU145 tumor cells with anti-TLR4 or anti-RAGE markedly abrogated sCLU induction and protective effect of the supernatant. Mechanistically, HMGB1 activated NFκB to promote sCLU gene expression and prevented the translocation of activated Bax to mitochondria to block cell death. Importantly, multiple currently-used chemotherapeutic drugs could release HMGB1 from tumor cells. These results suggest that acquisition of chemoresistance may involve the HMGB1/TLR4-RAGE/sCLU pathway triggered by dying cells to provide survival advantage to remnant live tumor cells.

Bone marrow mononuclear cells up-regulate toll-like receptor expression and produce inflammatory mediators in response to cigarette smoke extract.

Several reports link cigarette smoking with leukemia. However, the effects of cigarette smoke extract (CSE) on bone marrow hematopoiesis remain unknown. The objective of this study was to elucidate the direct effects of cigarette smoke on human bone marrow hematopoiesis and characterize the inflammatory process known to result from cigarette smoking. Bone marrow mononuclear cells (BMCs) from healthy individuals when exposed to CSE had significantly diminished CFU-E, BFU-E and CFU-GM. We found increased nuclear translocation of the NF-κB p65 subunit and, independently, enhanced activation of AKT and ERK1/2. Exposure of BMCs to CSE induced IL-8 and TGF-ß1 production, which was dependent on NF-κB and ERK1/2, but not on AKT. CSE treatment had no effect on the release of TNF-α, IL-10, or VEGF. Finally, CSE also had a significant induction of TLR2, TLR3 and TLR4, out of which, the up-regulation of TLR2 and TLR3 was found to be dependent on ERK1/2 and NF-κB activation, but not AKT. These results indicate that CSE profoundly inhibits the growth of erythroid and granulocyte-macrophage progenitors in the bone marrow. Further, CSE modulates NF-κB- and ERK1/2-dependent responses, suggesting that cigarette smoking may impair bone marrow hematopoiesis in vivo as well as induce inflammation, two processes that proceed malignant transformation.

Icariin and its derivative, ICT, exert anti-inflammatory, anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions.

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.