Characterization of the nature of granulocytic myeloid-derived suppressor cells in tumor-bearing mice

Related CCR Translation from CpG Blocks Immunosuppression by Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

Related CCR Translation from CpG Blocks Immunosuppression by Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

Biphasic development of splenic suppressor cells in tumor-bearing mice

Myeloid-derived suppressor cells (MDSC) is a group of cells with potent immune suppressive activity that accumulate in many pathologic conditions. In cancer, most of MDSC are represented by cells with granulocytic phenotype, CD11b+Ly6ClowLy6G+, and morphology and are thus called granulocytic MDSC (G-MDSC). This phenotype and morphology are also shared by mature neutrophils (Neu) in naive mice. The relationship, if any, between G-MDSC and Neu remains unclear. In this study, we compared tumor associated G-MDSC and mature Neu. G-MDSC were freshly isolated from spleen of tumor-bearing mice. Because there are no known specific markers that differentiate between Neu and G-MDSC in tumor being mice or Neu and immature myeloid cells in naïve mice; Neu were isolated from naive mice by mobilizing them to the peritoneum (pNeu). We found that in contrast to pNeu, a substantial proportion of G-MDSC express the M-CSF receptor, CD115, CXCR4 and the CD244 molecule, also known as 2B4. pNeu have high amounts of lysosomes, endosomes and proteasomes, which was validated using immunofluorescence microscopy to measure the expression of associated proteins, LAMP2, EEA1, PSMA5, respectively. G-MDSC had significantly lower expression of LAMP2, EEA1, PSMA5. Also, G-MDSC, compared to pNeu, had significantly lower phagocytic activity, as measured by the uptake of latex beads in vitro. On the other hand, G-MDSC, but not pNeu, suppressed T-cell IFN-γ production upon antigen-specific stimulation. Interestingly, pNeu had higher arginase activity and nitric oxide production than G-MDSC, while G-MDSC had a significantly higher increase in reactive oxygen species levels upon PMA stimulation in vitro. This may explain the differences in suppressive activity. G-MDSC survived 3 days in culture, in the presence of GM-CSF. Within 24 hours in culture with GM-CSF, they became phenotypically and functionally similar to pNeu, with increased phagocytic capacity and LAMP2 expression and decreased expression of CD115, CXCR4, and CD244. These data suggest that, in cancer, G-MDSC may represent pathologically activated precursors of neutrophils. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2011-3647

<div>Abstract<p><b>Purpose:</b> The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice.</p><p><b>Experimental Design:</b> We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors.</p><p><b>Results:</b> CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G<sup>hi</sup> polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G<sup>hi</sup> MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation <i>in vitro</i> and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity.</p><p><b>Conclusions:</b> We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process. <i>Clin Cancer Res; 17(7); 1765–75. ©2011 AACR</i>.</p></div>

The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice. We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors. CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G(hi) polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G(hi) MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation in vitro and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity. We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process.

<div>Abstract<p><b>Purpose:</b> The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice.</p><p><b>Experimental Design:</b> We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors.</p><p><b>Results:</b> CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G<sup>hi</sup> polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G<sup>hi</sup> MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation <i>in vitro</i> and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity.</p><p><b>Conclusions:</b> We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process. <i>Clin Cancer Res; 17(7); 1765–75. ©2011 AACR</i>.</p></div>

Metformin, an oral medicine broadly used for the treatment of type 2 diabetes, has been found to significantly improve tumor incidence and survival in large-scale clinical analysis. In recent years, the antitumor effect and mechanism of metformin have received much attention. Myeloid-derived suppressor cells (MDSCs), a major immunosuppressive cell type that accumulates in tumor-bearing hosts, can inhibit T cells and promote tumor immune escape. The mechanism by which metformin exerts its anti-tumor effect by regulating MDSCs remains unclear. Here, we found that metformin could inhibit the accumulation and suppressive capacity of G-MDSCs, delay tumor progression and elicit Th1 and CTL responses in murine colon cancer CT-26 cell-transplanted mice. In additionally, metformin could enhance the phosphorylation of AMPK, reduce STAT3 phosphorylation levels, and down-regulate the inhibitory function of G-MDSCs in vitro. These results suggest that metformin may be a potential clinical benefit for antitumor immunotherapy in tumor-bearing mice.

BackgroundMyeloid-derived suppressor cells (MDSCs) participate in tumor-elicited immunosuppression by dramatically blocking T-cell-induced antitumor responses, thereby influencing the effectiveness of cancer immunotherapies. Treatments that alter the differentiation and function of MDSCs can partially restore antitumor immune responses. The long noncoding RNA plasmacytoma variant translocation 1 (lncRNA Pvt1) is a potential oncogene in a variety of cancer types. However, whether lncRNA Pvt1 is involved in the regulation of MDSCs has not been thoroughly elucidated to date.MethodsMDSCs or granulocytic MDSCs (G-MDSCs) were isolated by microbeads and flow cytometry. Bone marrow derived G-MDSCs were induced by IL-6 and GM-CSF. The expression of lncRNA Pvt1 was measured by qRT-PCR. Specific siRNA was used to knockdown the expression of lncRNA Pvt1 in G-MDSCs.ResultsIn this study, we found that knockdown of lncRNA Pvt1 significantly inhibited the immunosuppressive function of G-MDSCs in vitro. Additionally, lncRNA Pvt1 knockdown reduced the ability of G-MDSCs to delay tumor progression in tumor-bearing mice in vivo. Notably, lncRNA Pvt1 was upregulated by HIF-1α under hypoxia in G-MDSCs.ConclusionsTaken together, our results demonstrate a critical role for lncRNA Pvt1 in regulating the immunosuppression activity of G-MDSCs, and lncRNA Pvt1 might thus be a potential antitumor immunotherapy target.

IFN-γ producing T cells contribute to the increase of myeloid derived suppressor cells in tumor-bearing mice after cyclophosphamide treatment

Recent studies show that the liver is a preferred organ for the accumulation of MDSC. In this study, we examined the effect of systemic RB6-8C5 treatment on hepatic MDSC in tumor-bearing mice. EL4 tumor-bearing mice were injected i.p. with RB6-8C5, and hepatic, splenic, and blood MDSCs were analyzed by flow cytometry. Unexpectedly, hepatic MDSC remained in the liver, although RB6-8C5 completely eliminated them from the spleen and peripheral blood 24 h after treatment. Secondary antibody staining confirmed the presence of RB6-8C5-bound MDSC in the liver of mice with s.c. tumors. Similar observations were made in two other (colon and melanoma) tumor models. Whereas RB6-8C5 injection induced cell death of hepatic MDSC, as shown by Annexin V/7-AAD staining, these cells were replaced immediately, leading to a constant, increased frequency of hepatic MDSC. Adoptively transferred MDSC migrated preferentially to the liver after RB6-8C5 treatment, suggesting that hepatic MDSCs are reconstituted rapidly after depletion. Finally, hepatic MDSC remained immunosuppressive despite RB6-8C5 injection. Our study demonstrates that RB6-8C5 is not suitable for depletion of hepatic MDSCs and analysis of their function.

Cancer immune evasion is an emerging hallmark of disease progression. Functional studies to understand the role of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment however, are limited by the lack of available specific cell surface markers. We adapted a competitive peptide phage display platform to identify candidate peptides binding MDSC specifically and generated peptide-Fc fusion proteins (peptibody). In multiple tumor models peptibody injection iv completely depleted blood, splenic, and intratumoral MDSC in tumor-bearing mice, without affecting proinflammatory immune cell types, such as dendritic cells. While control Gr-1 antibody depleted primarily granulocytic MDSC, peptibodies depleted both granulocytic and monocytic subsets. Remarkably, peptibody treatment was associated with inhibition of tumor growth in vivo, which was superior to Gr-1. Immunoprecipitation of MDSC membrane proteins identified S100 family proteins as candidate targets. Our strategy may be useful to identify novel diagnostic and therapeutic surface targets on rare cell subtypes, including human MDSC.

Accumulation of Myeloid-Derived Suppressor Cells in Bone Marrow in Multiple Myeloma Induces Tumor-Specific Immune Suppression and Promotes Tumor Growth

<p>Supplementary Figures S1-S5.</p>

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells which accumulate in stress conditions such as infection and tumor. Astaxanthin (ATX) is a well-known antioxidant agent and has a little toxicity. It has been reported that ATX treatment induces antitumor effects via regulation of cell signaling pathways, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2) signaling. In the present study, we hypothesized that treatment with ATX might induce maturation of MDSCs and modulate their immunosuppressive activity. Both in vivo and in vitro treatment with ATX resulted in up-regulation of surface markers such as CD80, MHC class II, and CD11c on both polymorphonuclear (PMN)-MDSCs and mononuclear (Mo)-MDSCs. Expression levels of functional mediators involved in immune suppression were significantly reduced, whereas mRNA levels of Nrf2 target genes were increased in ATX-treated MDSCs. In addition, ATX was found to have antioxidant activity reducing reactive oxygen species level in MDSCs. Finally, ATX-treated MDSCs were immunogenic enough to induce cytotoxic T lymphocyte response and contributed to the inhibition of tumor growth. This demonstrates the role of ATX as a regulator of the immunosuppressive tumor environment through induction of differentiation and functional conversion of MDSCs.