Skewing the Th cell phenotype toward Th1 alters the maturation of tumor-infiltrating mononuclear phagocytes.

Mononuclear phagocytes (MPCs) at the tumor site can be divided into subclasses, including monocyte-lineage myeloid-derived suppressor cells (MDSCs) and the immunosuppressive tumor-infiltrating macrophages (TIMs). Cancer growth coincides with the expansion of MDSCs found in the blood, secondary lymphoid organs, and tumor tissue. These MDSCs are thought to mature into macrophages and to promote tumor development by a combination of growth-enhancing properties and suppression of local antitumor immunoresponses. As little is known about either subset of MPCs, we investigated MPCs infiltrating into murine adenocarcinoma MCA38 tumors. We found that these MPCs displayed immunosuppressive characteristics and a MDSC cell-surface phenotype. Over 70% of the MPCs were mature (F4/80(+)Ly6C(-)) macrophages, and the rest were immature (F480(+) Ly6C(+)) monocytes. MPC maturation was inhibited when the cells infiltrated a tumor variant expressing IL-2 and soluble TNF type II receptor (sTNFRII). In addition, the IL-2/sTNFRII MCA38 tumor microenvironment altered the MPC phenotype; these cells did not survive culturing in vitro as a result of Fas-mediated apoptosis and negligible M-CSFR expression. Furthermore, CD4(+) tumor-infiltrating lymphocytes (TILs) in wild-type tumors robustly expressed IL-13, IFN-gamma, and GM-CSF, and CD4(+) TILs in IL-2/sTNFRII-expressing tumors expressed little IL-13. These data suggest that immunotherapy-altered Th cell balance in the tumor microenvironment can affect the differentiation and maturation of MPCs in vivo. Furthermore, as neither the designation MDSC nor TIM can sufficiently describe the status of monocytes/macrophages in this tumor microenvironment, we believe these cells are best designated as MPCs.

Tumor-infiltrating myeloid-derived suppressor cells are pleiotropic-inflamed monocytes/macrophages that bear M1- and M2-type characteristics.

Here, tumor-infiltrating CD11b(+) myelomonocytoid cells in murine colon adenocarcinoma-38 and GL261 murine glioma were phenotypically characterized. Over 90% were of the CD11b(+)F4/80(+) monocyte/macrophage lineage. They also had a myeloid-derived suppressor cell (MDSC) phenotype, as they suppressed the proliferation of activated splenic CD8(+) T cells and had a CD11b(+)CD11c(+)Gr-1(low)IL-4Ralpha(+) phenotype. In addition, the cells expressed CX(3)CR1 and CCR2 simultaneously, which are the markers of an inflammatory monocyte. The MDSCs expressed CD206, CXCL10, IL-1beta, and TNF-alpha mRNAs. They also simultaneously expressed CXCL10 and CD206 proteins, which are typical, classical (M1) and alternative (M2) macrophage activation markers, respectively. Peritoneal exudate cells (PECs) strongly expressed CD36, CD206, and TGF-beta mRNA, which is characteristic of deactivated monocytes. The MDSCs also secreted TGF-beta, and in vitro culture of MDSCs and PECs with anti-TGF-beta antibody recovered their ability to secrete NO. However, as a result of secretion of proinflammatory cytokines, MDSCs could not be categorized into deactivated monocyte/macrophages. Thus, tumor-infiltrating MDSCs bear pleiotropic characteristics of M1 and M2 monocytes/macrophages. Furthermore, CD206 expression by tumor-infiltrating MDSCs appears to be regulated by an autocrine mechanism that involves TGF-beta.

TNF expressed by tumor-associated macrophages, but not microglia, can eliminate glioma.

It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF.

Depletion of CD4+CD25+ regulatory T cells enhances interleukin-2-induced antitumor immunity in a mouse model of colon adenocarcinoma.

Interleukin 2 (IL)-2 induces antitumor immunity and clinical responses in melanoma and renal cell carcinoma. However, IL-2 also increases the number of CD4(+)CD25(+) regulatory T (Treg) cells that suppress antitumor immune responses. The aim of the present study was to elucidate the effect of depletion of Treg cells on IL-2-induced antitumor immunity. IL-2-transfected mouse colon adenocarcinoma (MC38/IL-2) cells were implanted subcutaneously or intrahepatically into male C57BL/6 mice, and tumor growth and the proportion of tumor-infiltrating lymphocytes with Treg-cell depletion in response to treatment with anti-CD25 monoclonal antibody (PC61) were determined. In mice treated with phosphate-buffered saline, 40-60% of MC38/IL-2 tumors were rejected. In contrast, all MC38/IL-2 tumors were rejected in mice treated with PC61. The number of tumor-infiltrating CD8(+) T cells in mice treated with PC61 was approximately twice that in mice treated with PBS. The numbers of tumor-infiltrating CD4(+) and natural killer cells were also increased significantly. To test the antimetastatic effects of IL-2 treatment in combination with Treg-cell depletion, human recombinant IL-2 (rIL-2) and PC61 were administered to mice implanted with MC38/mock cells in the spleen, and hepatic metastasis was investigated. The average liver weight in mice treated with rIL-2 plus PC61 was 1.04 +/- 0.03 g, less than that in mice treated with rIL-2 (2.04 +/- 0.51 g) or PC61 alone (1.81 +/- 0.38 g). We conclude that IL-2-induced antitumor immunity is enhanced by Treg-cell depletion and is due to expansion of the tumor-infiltrating cytotoxic CD8(+) T-cell population.

Interleukin-2 augmented activation of tumor associated macrophage plays the main role in MHC class I in vivo induction in tumor cells that are MHC negative in vitro.

The contribution of tumor associated macrophage (TAM) to the induction of major histocompatibility complex (MHC) class I expression in vivo has not been reported precisely. In this study, we utilized Interleukin-2 (IL-2) cDNA-introduced B16 melanoma cells (B16/IL-2) and vehicle-alone control cells (B16/mock) to examine whether TAM could contribute to the induction of MHC class I on B16 cells in vivo. Interestingly, although B16/mock and B16/IL-2 did not express MHC class I in vitro, MHC class I was strongly expressed in vivo in B16/IL-2 in comparison to B16/mock. Although in vivo treatment of anti-NK1.1 antibody abolished MHC expression in B16/mock in vivo, the same treatment did not influence MHC expression in B16/IL-2. Interestingly, both anti-asialo GM1 and anti-CD11b treatment strongly decreased MHC expression in B16/IL-2. TAM expressed both asialo GM1 and CD11b antigen, and TAM recovered from B16/IL-2 produced interferon gamma (IFNgamma) 6 times more than that from B16/mock. In addition, TAM recovered from B16/IL-2 secreted 33.64 times more IFNgamma in response to in vitro administration of IL-2. Therefore, we checked whether or not IL-2 could influence the expression of IL-2 receptors. TAM recovered from IL-2 expressed middle affinity receptor of IL-2 (CD122 and CD132) while that from B16/mock expressed low affinity receptor (CD25 and CD132). Finally, we observed that B16 cells became apoptotic with IFNgamma treatment in vitro. These results suggested that IL-2 augmented activation of TAM would play the main role in induction of the MHC class I molecule through secretion of IFNgamma, and would contribute to the IFNgamma-mediated apoptosis induction in tumor cells.

Preoperative radiotherapy contributes to induction of proliferative activity of CD8+ tumor-infiltrating T-cells in oral squamous cell carcinoma.

In order to evaluate host immune response to cancer, many methods have been applied. However, in the field of oral squamous cell carcinoma, evaluation of host immune response on the basis of proliferative activity of tumor-infiltrating T-cells (TIL) has not been reported. Therefore, we applied double immunohistochemical staining of proliferation markers Ki-67 and CD8 to surgically resected and paraffin-embedded tissue sections for 35 cases of oral squamous cell carcinoma. With this method, there was a significant correlation between the percentage of Ki-67+CD8+TIL and the intra-tumor epithelium infiltration rate of CD8+TIL (P=0.0237). In the process of analysis, we found that the proliferative activity of CD8+TIL tended to correlate (P=0.0859) with clinical N factor (lymph node metastasis), which was previously reported to suppress host immune response. We therefore assumed there was another factor inducing host immune response. The proliferative activity of CD8+TIL was well correlated with preoperative radiotherapy (P=0.0200) while there was no significant correlation between the proliferative activity of CD8+TIL and other clinical factors; age, tumor size, clinical stage, pathological N factor (P=0.5410, 0.7769, 0.1041, and 0.1072, respectively). Our present results strongly imply that preoperative radiotherapy is a very important factor in oral squamous cell carcinoma inducing host immune response regardless of the clinical factors present.

Analysis of ameloblastomas by comparative genomic hybridization and fluorescence in situ hybridization.

In order to characterize the chromosomal alterations in ameloblastomas, a combination of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) techniques was performed on 9 tumors. Chromosomal alterations including a gain at 1q and losses at 1pter, 10q, and 22q could be detected by CGH only in 1 tumor. Interphase FISH analysis, using centromeric probes for chromosomes 1, 10, and 22 as well as region-specific probes for 1p36 and 10q26, revealed the most frequent alterations to exist in the tumor with the abnormal CGH profile. These alterations included marked to slight increases of monosomic cells for chromosome 10 (91.5%), 10q26 (35.8%), 1p36 (24.4%), and chromosome 22 (18.8%), as well as significant elevations of trisomic cells for chromosome 1 (41.2%). Moreover, FISH analysis revealed a frequent loss of chromosome 22 in all tumors examined, except for one lesion, indicating that loss of the entire or a part of this chromosome is a common event in ameloblastomas, possibly being a predisposing factor to ameloblastoma tumorigenesis.

Natural killer cells play a role in MHC class I in vivo induction in tumor cells that are MHC negative in vitro.

The natural killer (NK) cell is one of the key cells in discriminating major histocompatibility complex (MHC) negative 'missing-self' target tumor cells, and interleukin 2 (IL-2) treatment was effective in inducing NK cell activation. In this study, we tried to clarify how poorly-immunogenic murine B16 melanoma could be discriminated in vivo by creating an IL-2 cDNA-transduced immunogene therapy model (B16/IL-2). In vitro study showed that IL-2 introduction did not induce MHC class I. However, immune cells depleted total tumor digest, which consisted of 90% anti-melanoma MM2-9B6-positive cells that revealed B16/IL-2 strongly, and control tumor cells (B16/mock) partially expressed MHC class I in vivo. In the B16/IL-2 model, NK cell infiltration was 10 times higher than B16/mock (7.6 versus 0.73, p=0.017). In addition, the cell surface of CD69-expressing NK cell population was increased in B16/IL-2, and the interferon gamma (IFNgamma) message level in NK cells was significantly increased in B16/IL-2 (p=0.0359). Interestingly, NK cell depletion in vivo completely abolished MHC class I expression on B16/mock, and decreased MHC class I expression and T-cell infiltration in B16/IL-2. These data suggest that NK cells are not only important for missing-self recognition, but are also crucial for induction of tumor cell MHC molecule expression and play an important role in helping acquired immunity to recognize tumor cells.

Interleukin-2 activated microglia engulf tumor infiltrating T cells in the central nervous system.

Interleukin-2 (IL-2) has been utilized to treat cancer patient. However, recent studies disclosed that IL-2 induces T cell death. To clarify IL-2 induced T cell apoptosis at tumor sites in the central nervous system (CNS), we utilized an intracranial implantation of IL-2 cDNA transduced murine tumor cells and examined freshly recovered tumor infiltrating T lymphocytes (TIL) with a magnetic beads separation method. CD8(+) TIL recovered from the IL-2 therapy model had three times more apoptosis than a control group, tumor weights at day 12 decreased (0.016 versus 0.041 g/mouse) and the number of TIL per gram of tumor tissue increase more than six times by IL-2 therapy (5.69x10(6) versus 33.7x10(7) cells per mouse). In addition, both activation marker expressions (CD25 and CD69) and cytokine message levels (interferon gamma, and tumor necrosis factor alpha) on CD8(+) TIL decrease in the IL-2 therapy model. Moreover, we detected higher CD8beta message levels in purified tumors associated with F4/80(+) cells from the IL-2 model than the control by a one-step RT-PCR method. Finally, we observed many CD8beta(+) TIL surrounded by numerous infiltrating F4/80(+) cells in the tumor tissues of the IL-2 therapy model by immuno-fluorescence microscopic analysis. Our data show that IL-2 sensitization of apoptosis induction for CD8(+) TIL occurred and the apoptotic T cells were eliminated by F4/80(+) microglia in the CNS. Moreover, this is the first report describing in situ elimination of TIL by F4/80(+) phagocytic cells in the CNS.

In situ detection of O6-methylguanine-DNA methyltransferase messenger RNA in paraffin-embedded human astrocytic tumor tissues by nested in situ RT-PCR is useful in predicting chemotherapy-resistance of tumors.

O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that interferes with chemotherapeutic effect of alkylating agents. We performed in situ detection of MGMT mRNA utilizing the nested RT-PCR method in tissue sections (nested in situ RT-PCR). We analyzed 34 samples of paraffin-embedded astrocytic tumor tissue sections with this method [3 astrocytomas, 14 anaplastic astrocytomas (AA), and 17 glioblastoma multiformes (GBM)]. Twenty-five cases (73.5% of all cases) were positive for MGMT either with our method or immunohistochemistry (IHC). Moreover, with our method >25% of the cells in the tumor tissue expressed MGMT in contrast to >4% with IHC among the population of MGMT positive cases. Our method was significantly more sensitive than IHC (p=0.0004). The present results suggest that potentially there is a greater population of MGMT positive cells in astrocytic tumor tissues than the one evaluated with IHC. These findings suggest that the >25% of the MGMT positive cells are involved in the interference with the chemotherapeutic effect of alkylating agents. The MGMT expressing cell population was markedly decreased in GBM compared with AA (26.1% vs 62.1%). The main reason for this marked decrease was that MGMT was expressed in only 9 of 17 cases of GBM in contrast to all AA cases that expressed MGMT. This result suggests that there are potentially two populations of GBM on the basis of MGMT expression, in which the negative population might be mainly composed of de novo GBM. Therefore, it is suggested that our method is practically useful to detect any drug resistance gene product with high sensitivity and would provide a chance to evaluate the chemotherapeutic effect of any agents in an individual patient based manner.