Most malignant tumors evidence infiltration of many macrophages. In this study, we investigated an anti‐inflammatory macrophage phenotype (M2) in clear cell renal cell carcinoma (RCC) using CD163 and CD204 as markers. Immunostaining showed a correlation between the number of CD163+ cells and age, sex, nuclear grade, and TNM classification. High infiltration of CD163+ cells was significantly associated with poor clinical prognosis in univariate analysis but not in multivariate analysis. We also carried out in vitro studies to examine cell–cell interactions between macrophages and cancer cells. Culture supernatants from RCC cell lines induced polarization of macrophages toward the M2 phenotype. Coculture of macrophages with cancer cells significantly induced activation of signal transducers and activators of transcription‐3 (Stat3) in the cancer cells. Direct coculture of RCC cells with macrophages led to stronger activation of Stat3 in the cancer cells than did indirect coculture using Transwell chamber dishes. Because RCC cells expressed membrane‐type macrophage colony‐stimulating factor (mM‐CSF) on the cell surface, we suggested that this mM‐CSF plays an important role in direct cell–cell interactions. Stat3 activation in cancer cells that was induced by coculture with macrophages was suppressed by downregulation of the M‐CSF receptor (M‐CSFR) in macrophages and by an inhibitor of M‐CSFR. In conclusion, investigation of CD163+ tumor‐associated macrophages would be useful for assessment of the clinical prognosis of patients with ccRCC. Cell–cell interactions mediated by mM‐CSF and M‐CSFR binding could contribute to cancer cell activation. (Cancer Sci 2011; 102: 1424–1431)

Macrophage colony-stimulating factor (M-CSF) regulates the production, survival and function of macrophages through Fms, the receptor tyrosine kinase. Recently, interleukin-34 (IL-34), which shares no sequence homology with M-CSF, was identified as an alternative Fms ligand. Here, we provide the first evidence that these ligands indeed resemble but are not necessarily identical in biological activity and signal activation. In culture systems tested, IL-34 and M-CSF showed an equivalent ability to support cell growth or survival. However, they were different in the ability to induce the production of chemokines such as MCP-1 and eotaxin-2 in primary macrophages, the morphological change in TF-1-fms cells and the migration of J774A.1 cells. Importantly, IL-34 induced a stronger but transient tyrosine phosphorylation of Fms and downstream molecules, and rapidly downregulated Fms. Even in the comparison of active domains, these ligands showed no sequence homology including the position of cysteines. Interestingly, an anti-Fms monoclonal antibody (Mab) blocked both IL-34-Fms and M-CSF-Fms binding, but another MAb blocked only M-CSF-Fms binding. These results suggested that IL-34 and M-CSF differed in their structure and Fms domains that they bound, which caused different bioactivities and signal activation kinetics/strength. Our findings indicate that macrophage phenotype and function are differentially regulated even at the level of the single receptor, Fms.

Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec–induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor–sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec− T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type–specific manner.

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program.

We previously showed tumor-associated macrophages/microglia (TAMs) polarized to the M2 phenotype were significantly involved in tumor cell proliferation and poor clinical prognosis in patients with high grade gliomas. However, the detailed molecular mechanisms involved in the interaction between TAMs and tumor cells have been unclear. Current results reveal that, in coculture with human macrophages, BrdU incorporation was significantly elevated in glioma cells, and signal transducer and activator of transcription-3 (Stat3) activation was found in both cell types. Direct mixed coculture led to stronger Stat3 activation in tumor cells than did indirect separate coculture in Transwell chamber dishes. Screening with an array kit for phospho-receptor tyrosine kinases revealed that phosphorylation of macrophagecolony stimulating factor receptor (M-CSFR, CD115, or c-fms) is possibly involved in this cell-cell interaction; M-CSFR activation was detected in both cell types. Coculture-induced tumor cell activation was suppressed by siRNA-mediated downregulation of the M-CSFR in macrophages and by an inhibitor of M-CSFR (GW2580). Immunohistochemical analysis of phosphorylated (p) M-CSFR, pStat3, M-CSF, M2 ratio, and MIB-1(%) in high grade gliomas revealed that higher staining of pM-CSFR in tumor cells was significantly associated with higher M-CSF expression and higher MIB-1(%). Higher staining of pStat3 was associated with higher MIB-1(%). High M2 ratios were closely correlated with high MIB-1(%) and poor clinical prognosis. Targeting these molecules or deactivating M2 macrophages might be useful therapeutic strategies for high grade glioma patients. (Cancer Sci 2012; 103: 2165-2172

No abstract

Clear cell renal cell carcinoma (ccRCC) is one of most common cancers in urogenital organs. Although recent experimental and clinical studies have shown the immunogenic properties of ccRCC as illustrated by the clinical sensitivities to various immunotherapies, the detailed immunoregulatory machineries governing the tumorigenicity of human ccRCC remain largely obscure. In this study, we demonstrated the clinical significance and functional relevance of T-cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) expressed on tumor cells and myeloid cells in patients with ccRCC. TIM-3 expression was detected on cancer cells and CD204 þ tumor-associated macrophages (TAM), and higher expression level of TIM-3 was positively correlated with shorter progression-free survival (PFS) in patients with ccRCC. We found that TIM-3 expression was detected on a large number of tumors, and there was significant correlation between an increased number of TAMs and high expression level of TIM-3 in patients with ccRCC. Furthermore, TIM-3 rendered RCC cells with the ability to induce resistance to sunitinib and mTOR inhibitors, the standard regimen for patients with ccRCC, as well as stem cell activities. TIM-3 expression was induced on CD14

An animal model in which the human immune system can be reconstituted is necessary to study acquired immunity in vivo. We report here a novel model, the NOD/SCID/JAK3(null) mouse, for the human immune system's development. Newborn mice transplanted with human cord blood CD34(+) cells intrahepatically, developed human T and B cells, and myeloid and plasmacytoid dendritic cells. The T and B cells had a naïve to memory phenotype, and included plasma cells. The human acquired immune system can be reconstituted from CD34(+) cells in NOD/SCID/JAK3(null) mice. This model is a powerful tool for the study of human immunity.