Diana M. Lopez

The Liver is a Site for Tumor Induced Myeloid-Derived Suppressor Cell Accumulation and Immunosuppression

Tumor-induced immunosuppression plays a key role in tumor evasion of the immune system. A key cell population recognized as myeloid-derived suppressor cells (MDSC) contributes and helps orchestrate this immunosuppression. MDSC can interact with T cells, macrophages, and natural killer cells to create an environment favorable for tumor progression. In various tumor models, their presence at high levels has been reported in the bone marrow, blood, spleen, and tumor. We report for the first time that MDSC accumulate and home to the liver in addition to the other organs. Liver MDSC suppress T cells and accumulate to levels comparable with splenic MDSC. Additionally, hematopoiesis in the liver contributes to the dramatic expansion of MDSC in this organ. Furthermore, MDSC in the liver interact with macrophages, also known as Kupffer cells, and cause their up-regulation of PD-L1, a negative T-cell costimulatory molecule. The liver is thus an organ in which MDSC accumulate and can contribute to immunosuppression directly and indirectly. MDSC play a role in various pathologic states in addition to cancer, and these results contribute to our understanding of their biology and interactions with immune-related cells.

Identification of a Subpopulation of Macrophages in Mammary Tumor–Bearing Mice That Are Neither M1 nor M2 and Are Less Differentiated

Systemic and local immune deficiency is associated with cancer, and the role of M2 tumor-associated macrophages in this phenomenon is well recognized. However, the immune status of macrophages from peripheral compartments in tumor hosts is unclear. Peritoneal macrophages (PEM) are derived from circulating monocytes and recruited to the peritoneal cavity where they differentiate into macrophages. We have previously shown that PEMs from mice bearing D1-DMBA-3 mammary tumors (T-PEM) are deficient in inflammatory functions and that this impairment is associated with diminished expression of transcription factors nuclear factor kappaB and CAAT/enhancer-binding protein. We now provide evidence that T-PEMs display neither M1 nor M2 phenotypes, yet exhibit deficiencies in the expression of several inflammatory cytokines and various proinflammatory signaling pathways. Moreover, due to nuclear factor kappaB down-regulation, increased apoptosis was observed in T-PEMs. We report for the first time that macrophage depletion is associated with increased macrophage progenitors in bone marrow. Furthermore, T-PEMs have a lower expression of macrophage differentiation markers F4/80, CD68, CD115, and CD11b, whereas Gr-1 is up-regulated. Our results suggest that T-PEMs are less differentiated and represent a newly derived population from blood monocytes. Lastly, we show that transforming growth factor-beta and prostaglandin E(2), two immunosuppressive tumor-derived factors, may be involved in this phenomenon.

Splenic macrophages from tumor-bearing mice co-expressing MAC-1 and MAC-2 antigens exert immunoregulatory functions via two distinct mechanisms

Tumor burden has been shown to induce a variety of phenotypic and functional changes in the cellular constituents of the hosts immune system. These changes have been implicated as mechanisms by which tumors avoid rejection. Studies of BALB/c mice bearing a D1‐DMBA‐3 mammary adenocarcinoma showed alterations of the splenocyte populations. There was a five‐fold increase of macrophages (Mφ) that were phenotypically and functionally analyzed to establish their role in tumor‐induced modifications of the hosts immune response. Monoclonal antibody staining defined a Mac‐1+2+ population which comprised up to 20% of the splenocytes in tumor‐bearers (TB), but is negligible in spleens from normal mice. These Mao‐1+2+ Mφ were found to mediate down‐regulation of both polyclonal and antigen‐specific T and B cell responses in vitro and in vivo. Although B cell responses were suppressed via prostaglandin E2 (PGE2) production by the TB Mφ, T cell responses were relatively refractory to PGE2‐mediated down‐regulation. Instead, they were suppressed by a contact‐dependent T cell‐Mφ interaction. Furthermore, tumor‐derived factors such as granulocyte‐Mφ colony‐stimulating factor (GM‐CSF) seem to play an important role in the induction and expansion of the Mac‐1+2+ Mφ. These cells appear to mediate down‐regulation of the host immune responses by at least two distinct mechanisms: 1) PGE2 production and 2) a cell contact‐dependent, but non‐major‐histocompatibility‐complex‐specific, interaction.

Up-Regulation of Matrix Metalloproteinase-9 in T Lymphocytes of Mammary Tumor Bearers: Role of Vascular Endothelial Growth Factor

Matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme, is crucial in tumor invasion and metastasis and is implicated in leukocyte extravasation. In this report, we demonstrate that during growth of the D1–7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is progressive up-regulation of MMP-9 in splenic T cells at both the transcriptional and translational levels. Our previous work has identified several factors produced by this tumor, including PGE2, GM-CSF, and phosphatidyl serine; however, none of these agents induces increased production of MMP-9 by normal splenic T cells. Although not produced by the tumor, TNF-α and IL-6 are up-regulated in both macrophages and B cells in tumor-bearing mice. Exposure of normal T cells to these two cytokines, however, also fails to up-regulate MMP-9 production. Vascular endothelial growth factor (VEGF) is produced by many tumors, and we determined that the mammary tumor used in our studies expresses high levels of this angiogenic growth factor. Importantly, splenic T cells from tumor bearers constitutively produce increased amounts of VEGF, and treatment of normal T cells with VEGF results in up-regulated MMP-9 production. Of crucial importance is the finding that tumor-infiltrating T cells also produce high levels of VEGF and MMP-9. Our studies indicate that VEGF can act directly on T lymphocytes and that elevated VEGF levels may contribute to the aberrant MMP-9 secretion by mammary tumor bearers’ T cells.

Immune modulation by melanoma‐derived factors

Abstract:  Melanomas, while the less common of skin cancers, are highly aggressive and once they metastasize usually indicate a poor prognosis. Melanomas are in many cases immunogenic and thus have been a prime target for immunotherapy, which has resulted in objective responses in some patients. To understand why antitumor immunity fails, and for the purpose of discovering new targets to improve therapy, there has been great interest to analyse the antitumor immune responses which exist in these patients, and uncover mechanisms which block tumor‐specific immune responses. It is now evident that immunosuppressive cell networks and factors play a major role in the failure of the antitumor immune responses and therapies to eradicate the tumor. In this review, the factors produced by melanomas which can modulate and enhance these suppressive mechanisms are discussed. The roles of immature dendritic cells, neutrophils, T‐regulatory cells, myeloid‐derived suppressor cells and M2 macrophages or tumor‐associated macrophages are described. Furthermore, taking into consideration of the cross‐talk which exists among these different cell types and the cycle of immunosuppression which is evident in melanoma cancer patients and animal models, will be important for future therapeutic approaches.

Phylogenetic studies on T cells. I. Lymphocytes of the shark with differential response to phytohemagglutinin and concanavalin A

Abstract Peripheral blood lymphocytes of the nurse shark ( Ginglymostoma cirratum ) respond to stimulation by concanavalin A (Con A) as evidenced by increased incorporation of tritiated thymidine. Separation by means of Ficoll-Isopaque yields two or more bands and a sediment, all of which contain lymphocytes responsive to Con A. Only the bottom cells react to phytohemagglutinin (PHA). This reaction cannot be detected in the unseparated lymphocyte population. Thus, only a unique subset of lymphocytes appears to be responsive to PHA and is probably blocked in its response by other cells. The findings suggest that differentiation toward Con A responsiveness may have preceded phylogenetically the responsiveness to PHA. Judging by the requirement for high concentrations of both mitogens the receptor sites on shark lymphocytes appear to be present in lower densities than on lymphocytes of higher vertebrates.

Early Block in Maturation Is Associated with Thymic Involution in Mammary Tumor-Bearing Mice

We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5′-bromo-2′-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4−8− thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4−8− thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4−8− thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.

Urokinase-mediated recruitment of myeloid-derived suppressor cells and their suppressive mechanisms are blocked by MUC1/sec

The transmembrane isoform of mucin 1 (MUC1/TM) is a well-recognized tumor antigen, contributing to tumorigenesis and immune evasion. Although MUC1/TM has been correlated with malignancy, we have previously reported on antitumor properties and prevention of tumor development by a secreted splice variant of MUC1 (MUC1/sec). Because myeloid-derived suppressor cells (MDSCs) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by tumor cells expressing either MUC1/TM or MUC1/sec. DA-3 tumor cells expressing MUC1/sec recruit dramatically lower levels of MDSCs, relative to MUC1/TM-expressing DA-3 cells. Because MUC1/sec was previously shown to down-regulate tumor expression of urokinase plasminogen activator (uPA), a protease linked to tumor aggressiveness and metastasis, the potential role of uPA in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSCs, and correlates with tumor development. In addition to diminishing recruitment of MDSCs, the effect of MUC1/sec on MDSC-suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide, is capable of blocking expression of arginase 1 and production of reactive oxygen species in MDSCs, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting MDSCs.

MUC1/sec-Expressing Tumors Are Rejected In Vivo by a T Cell-Dependent Mechanism and Secrete High Levels of CCL2

MUC1/sec is a secreted form of the glycoprotein mucin 1 (MUC1). To characterize the role that MUC1 and MUC1/sec have in tumor progression, these genes were expressed in DA-3 mammary tumor cells. DA-3 cells and DA-3 cells expressing the transmembrane MUC1 gene (DA-3/TM) grow with similar kinetics in BALB/c mice. Surprisingly, DA-3 cells expressing and secreting MUC1/sec (DA-3/sec) fail to form tumors in vivo. The mechanism of rejection was evaluated using mice deficient in constituents of the immune system. All mice lacking IFN-γ, NK, NKT, or macrophages formed DA-3/sec tumors that regressed shortly after implantation. However, progressively growing DA-3/sec tumors developed in mice devoid of T lymphocytes. The importance of T lymphocytes in the rejection of DA-3/sec tumors was further supported by detection of DA-3-specific CTL in mice challenged with the DA-3/sec tumor. Recruitment of appropriate APC and effector cells is an important first step in the tumor clearance. Indeed, DA-3/sec cells or cell supernatants recruited 3–4 times as many macrophages as DA-3/TM cells in vivo, suggesting that a secreted chemotactic product is produced from DA-3/sec cells. RNA and protein analysis of DA-3/sec cells revealed that several genes are up-regulated by MUC1/sec expression, including MCP-1 (CCL-2). These results suggest DA-3/sec cells are capable of recruiting immune cells, and that rejection of DA-3/sec tumors, although aided by cells of the innate immune response, is ultimately due to T cell-mediated events.

Downregulation of interleukin-7 and hepatocyte growth factor in the thymic microenvironment is associated with thymus involution in tumor-bearing mice

During mammary tumorigenesis, there is a profound thymic involution associated with severe depletion of the most abundant subset of thymocytes, CD4+CD8+ immature cells, and an early arrest in at least two steps of T cell differentiation. Thymic atrophy that is normally related with aging has been observed in other model systems, including graft-vs-host disease (GVHD) and tumor development. However, the mechanisms involved in this phenomenon remain to be elucidated. Vascular endothelial growth factor (VEGF) has been associated with thymic involution, when expressed at high levels systemically. In thymuses of D1-DMBA-3 tumor-bearing mice, this growth factor is diminished relative to the level of normal thymuses. Interestingly, the expression of hepatocyte growth factor (HGF), which has been associated with proliferation, cell survival, angiogenesis and B-cell differentiation, is profoundly down-regulated in thymuses of tumor bearers. In parallel, IL-7 and IL-15 mRNA, crucial cytokines involved in thymocytes development and cellular homeostasis, respectively, are also down-regulated in the thymuses of tumor hosts as compared to those of normal mice. Injection of HGF into mice implanted with mammary tumors resulted in normalization of thymic volume and levels of VEGF, IL-7 and IL-15. While, injections of IL-7 partially restored the thymic involution observed in the thymuses of tumor-bearing mice, injection of IL-15 did not have any significant effects. Our data suggest that the downregulation of HGF and IL-7 may play an important role in the thymic involution observed in tumor-bearing hosts.