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Immune escape and tolerance in the tumor microenvironment are closely involved in tumor progression, and are caused by T-cell exhaustion and mediated by the inhibitory signaling of immune checkpoint molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated protein 4, and T-cell immunoglobulin and mucin domaincontaining molecule-3. In the present study, we investigated the expression of the PD-1 ligand 1 (PD-L1) in a lymphoma microenvironment using paraffin-embedded tissue samples, and subsequently studied the detailed mechanism of upregulation of PD-L1 on macrophages using cultured human macrophages and lymphoma cell lines. We found that macrophages in lymphoma tissues of almost all cases of adult T-cell leukemia/lymphoma (ATLL), follicular lymphoma and diffuse large B-cell lymphoma expressed PD-L1. Cell culture studies showed that the conditioned medium of ATL-T and SLVL cell lines induced increased expression of PD-L1/2 on macrophages, and that this PD-L1/2 overexpression was dependent on activation of signal transducer and activator of transcription 3 (Stat3). In vitro studies including cytokine array analysis showed that IL-27 (heterodimer of p28 and EBI3) induced overexpression of PD-L1/2 on macrophages via Stat3 activation. Because lymphoma cell lines produced IL-27B (EBI3) but not IL-27p28, it was proposed that the IL-27p28 derived from macrophages and the IL-27B (EBI3) derived from lymphoma cells formed an IL-27 (heterodimer) that induced PD-L1/2 overexpression. Although the significance of PD-L1/2 expressions on macrophages in lymphoma progression has never been clarified, an IL-27-Stat3 axis might be a target for immunotherapy for lymphoma patients.

Cancer stem-like cells (CSC) or cancer-initiating cells are now considered to be an important cell population related to cancer recurrence and the resistance to anti-cancer therapy. Tumor-associated macrophages (TAM) are a main component of stromal cells and are related to cancer progression in clear cell renal cell carcinoma (ccRCC). Because the detailed mechanisms allowing the maintenance of CSC in cancer tissues remain unclear, we investigated the relationship between TAM and CD44-expressing cancer cells in ccRCC. CD44 was used as a marker for CSC, and CD163 and CD204 were used as markers for TAM. CD44-positive cancer cells were detected in 37 of the 103 cases. Although statistical analysis showed no relationship between CD44-positive cancer cells and the clinical course, the distribution of CD44-positive cancer cells was significantly associated with a high density of TAM. Our in vitro study using RCC cell lines and human macrophages demonstrated that CD44 expression was upregulated by direct co-culture with macrophages. Silencing of TNF-alpha on macrophages abrogated the upregulation of CD44 expression in cancer cells. Macrophage-induced CD44 overexpression was also suppressed by NF-κB inhibitors. These results suggest that TNF-alpha derived from TAM is linked to CD44 overexpression via NF-κB signaling in ccRCC.