TGF-β downregulates the activating receptor NKG2D on NK cells and CD8+ T cells in glioma patients

The activating receptor NKG2D, expressed by natural killer (NK) cells and CD8⁺ T cells, has a role in the specific killing of transformed cells. We examined NKG2D expression in patients with glioblastoma multiforme and found that NKG2D was downregulated on NK cells and CD8⁺ T cells. Expression of NKG2D on lymphocytes significantly increased following tumor resection and correlated with an increased ability to kill NKG2D ligand-positive tumor targets. Despite the presence of soluble NKG2D ligands in the sera of glioblastoma patients, NKG2D downregulation was primarily caused by tumor-derived tumor growth factor-β, suggesting that blocking of this cytokine may have therapeutic benefit.     

Decreased accumulation of immune regulatory cells is correlated to the antitumor effect of IFN-γ overexpression in the tumor

During mammary tumorigenesis, there is a profound tumor-induced immunosuppression and a progressive thymic atrophy associated with tumor development. IFN-γ has been shown to be effective in enhancing antitumor responses in several tumor models, however, how IFN-γ exerts its anti-tumor effect is largely controversial. In the present study we have used a mammary tumor model to investigate whether the levels of IFN-γ have an important role in the tumor-induced immuno-suppression as well as in the pathogenesis of the thymic atrophy. We evaluated this possibility using DA-3 cells transfected to express IFN-γ (DA-3/IFN-γ), a system that provides constant, local production of IFN-γ within the tumor microenvironment. Overexpression of IFN-γ in the mammary tumor results in a marked delay of tumor growth, a reduction in regulatory T cells and myeloid-derived suppressor cells accumulation mostly due to down-regulation of chemokines implicated in the recruitment of immune regulatory cells, and a blockage in the tumor-associated thymus atrophy. Collectively, our data suggest that the replacement of the faulty levels of IFN-γ in the tumor results in a diminution of the tumor-induced immune suppression caused by the mammary tumor development.     

CD8+CD28− cells and CD4+CD25+ regulatory T cells in the peripheral blood of advanced stage lung cancer patients

Aim To evaluate the CD8+CD28? and CD4+CD25+ regulatory T (Treg) cells in addition to other some lymphocyte subgroups in peripheral blood of advanced stage lung cancer patients. Methods The study group (n = 28) comprised chemotherapy and radiotherapy naïve patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The control group (n = 22) consisted of age- and sex-matched healthy volunteers. Flow cytometry was used to count T cells, natural killer (NK) cells and CD4+CD25 Treg cells, and for CD8+ T cell subgroup analysis. Flow cytometry was performed and annexin V binding was used for apoptotic cell evaluation. Results In patient group, the percentage of CD8+CD28? cells among lymphocytes was elevated, and there was also an increase in the CD28?/CD28+ cell ratio among CD8 lymphocyte population. The distribution of CD8 cells was different in lung cancer patients when compared with the control group. The absolute count of CD4+CD25^bright cells and the percentages of these cells among total lymphocytes were higher in the patient group. The Annexin V(+) cell percentages among CD8+CD28? and CD8+CD28+ lymphocytes were higher in the patient group than in the control group. No differences were found between the NSCLC and SCLC patients with respect to the hematological parameters and the distribution of lymphocyte subgroups. In NSCLC patients, the percentage of CD8+CD28? cells among the lymphocyte population was higher in patients with stage IV than those with stage III. Conclusion These findings may reflect the possibility of tumor-induced immunosuppression and they should be complemented with further studies.     

Decreased number and reduced NKG2D expression of Vδ1 γδ T cells are involved in the impaired function of Vδ1 γδ T cells in the tissue of gastric cancer

Background

In cancer patients, impaired function of immune cells—such as CD8⁺ T cells, NK cells, and dendritic cells—reportedly results in tumor progression. Although γδ T cells also play a critical role in tumor defense, their function remains unclear in cancer patients.

Methods

The frequency and function of γδ T cells in peripheral blood, normal gastric mucosa, and cancer tissue were evaluated by multicolor flow cytometry. We also determined NKG2D expression on γδ T cells in gastric cancer patients.

Results

The frequency of Vδ1 γδ T cells in gastric cancer tissue is significantly lower than in normal gastric mucosa; however, differences in the frequencies of Vδ2 and Vγ9 γδ T cells between normal gastric mucosa and gastric cancer tissue were not statistically significant. The Vδ1 γδ T cells from gastric cancer tissue produce significantly less IFN-γ than those from normal gastric mucosa do. Expression of NKG2D on Vδ1 γδ T cells from gastric cancer tissue was significantly lower than in normal gastric mucosa. We also found a significant correlation between NKG2D expression and IFN-γ production of Vδ1 γδ T cells in gastric cancer tissue.

Conclusion

Vδ1 γδ T cells show decreased frequency and impaired function in gastric cancer tissue, for which decreased NKG2D expression might be one of the mechanisms. Modalities specifically targeting NKG2D in Vδ1 γδ T cells may provide a breakthrough treatment for gastric cancer patients.     

Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.     

β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

Over-activation of SUMOylation is correlated with poor prognosis in multiple myeloma (MM), with the mechanism unclear. Wnt signaling is one of the aberrantly regulated pathways related to cancer tumorigenesis and progression. Whether SUMOylation is involved in regulating the activity of Wnt/β-catenin pathway, however, has not been reported in MM. Here we found that the TOPflash reporter activity and the expression of Wnt/β-catenin target genes can be down-regulated after interference with SUMOylation through SUMO-1 small interfering RNA (siRNA). SUMOylation inhibition down-regulated β-catenin at protein level via promotion of ubiquitin-proteasomal mediated degradation. Furthermore, over-expression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition, indicating that β-catenin was responsible for the observed effect on Wnt/β-catenin pathway. To gain a clearer view, we exploited the inter-protein interactions of β-catenin and SUMO-1 in myeloma cell lines. Immunoprecipitation and immunofluorescence assay proved that β-catenin is subjected to SUMOylation in vivo, which may, at least partially explain the impact of SUMOylation inhibition on β-catenin. The association of SUMO-1 and β-catenin was confirmed in myeloma patient samples. Taken together, our data proved that SUMOylation inhibition down-regulates Wnt/β-catenin pathway by promoting the ubiquitin-proteasomal mediated degradation of β-catenin. SUMOylation of β-catenin is part of the mechanisms involved in the dysregulated proliferation of myeloma cells.     

The Wnt/β-catenin signaling pathway is involved in the antitumor effect of fulvestrant on rat prolactinoma MMQ cells

Although an antiestrogen treatment for estrogen-dependent diseases, such as breast cancers, has been reported, the effect of this endocrine therapy on prolactinomas and its possible mechanism are unclear. This study investigates the antitumor effect of fulvestrant, which is a new estrogen receptor antagonist, on rat prolactinoma MMQ cells and the possible roles of the Wnt/β-catenin signaling pathway that is involved in this antitumor effect. To investigate the antitumor effect of fulvestrant, the effects of exposure to gradient doses of fulvestrant (0, 0.04, 1, 25, and 625 nM) on the proliferation of cells and the secretion of prolactin (PRL) were studied. Then, the expression levels of the Wnt/β-catenin signaling pathway-related proteins β-catenin and Wnt inhibitory factor-1 (WIF-1) were measured to investigate their possible roles in the antitumor effect of fulvestrant. The cells were also treated with decitabine (10 μM) to investigate the epigenetic mechanism of WIF-1 expression. The proliferation of MMQ cells and the secretion of PRL were suppressed by fulvestrant in a dose-dependent manner (up to 57.0?±?3.9 % and 51.2?±?4.9 %, respectively). β-Catenin expression was downregulated and was positively correlated with ER-α expression (P?<?0.01). As a tumor suppressor, WIF-1 expression was upregulated and was negatively correlated with ER-α expression (P?<?0.01). Furthermore, WIF-1 expression was upregulated via the hypomethylation of the promoter by decitabine, and cellular proliferation was correspondingly suppressed (37.8?±?4.3 %). Antitumor effect of fulvestrant was partially disrupted by SB 216763 via activation of the Wnt/β-catenin pathway. In conclusion, through the Wnt/β-catenin signaling pathway, fulvestrant can suppress the proliferation of MMQ cells and the secretion of PRL.     

Tumor-derived chemokine CCL5 enhances TGF-β-mediated killing of CD8(+) T cells in colon cancer by T-regulatory cells

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (T(reg)). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T(reg) infiltration and CD8(+) T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T(reg) against CD8(+) T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T(reg) cell infiltration and CD8(+) T-cell apoptosis was noted. TGF-β signaling blockade diminished apoptosis of CD8(+) T cells, implicating TGF-β as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-β by CCR5-deficient T(reg) or to enhance their cytotoxic effects against CD8(+) T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of T(reg) in inhibiting the antitumor responses of CD8(+) T cells, in the same way as CCL5 signaling blockade. Together, our findings establish that CCL5/CCR5 signaling recruits T(reg) to tumors and enhances their ability to kill antitumor CD8(+) T cells, thereby defining a novel mechanism of immune escape in colorectal cancer.     

Determination of a CD4+CD25−FoxP3+ T cells subset in tumor-draining lymph nodes of colorectal cancer secreting IL-2 and IFN-γ

CD4⁺CD25⁻FoxP3⁺ cells are a newly recognized subset of T cells which was first reported in autoimmune diseases. In our previous study, this subset was detected in tumor-draining lymph nodes (TDLNs) of patients with breast cancer. As little is known about their function in TDLNs of cancer patients, in this study, their frequency as well as their ability to produce interleukin (IL)-2, IL-10, or interferon (IFN)-γ were investigated in TDLNs of colorectal cancer (CRC) patients. Mononuclear cells were isolated from lymph nodes of 13 patients with CRC using Ficoll-Hypaque gradient centrifugation. Cells were stimulated in vitro and stained with CD25, CD4, FoxP3, IFN-γ, IL-10, and IL-2 or isotype matched antibodies and subjected to flow cytometry. The frequency of CD4⁺CD25⁻FoxP3⁺CD127^dim/− cells was significantly lower than CD4⁺CD25⁺FoxP3⁺CD127^dim/− population in TDLNs of CRC patients. The percentage of CD127^dim/− cells and also the MFI of FoxP3 expression was significantly lower in CD4⁺CD25⁻FoxP3⁺ in comparison with CD4⁺CD25⁺FoxP3⁺ population. Moreover, CD4⁺CD25⁻FoxP3⁺ cells contained higher percentages of IL-2- and IFN-γ-producing cells than CD4⁺CD25⁺FoxP3⁺ subpopulation. But, no difference was seen between two subsets in terms of IL-10 production. CD4⁺CD25⁻FoxP3⁺ cells in TDLNs of CRC patients had lower suppressive and higher effector properties in comparison with CD4⁺CD25⁺FoxP3⁺ conventional regulatory T cells.

     

HIF2α is involved in the expansion of CXCR4-positive cancer stem-like cells in renal cell carcinoma

Background:

Hypoxia and the subsequent activation of hypoxia-inducible factor-2α (HIF2α) contribute to the progression of a variety of cancers. However, their role in the generation of renal cell carcinoma-derived stem cells has not been fully addressed.

Methods:

A sphere formation assay, cell proliferation, RT–PCR, western blot, FACS, immunohistochemistry and tumour xenograft were used to study the role of HIF2α.

Results:

Propagation of four renal cell carcinoma (RCC) cell lines (Caki-1, Caki-2, 786-O, 769-P) in anchorage-independent floating spheres led to the expansion of cells bearing the CXCR4 (CD184) surface marker. Inhibition of the CXCR4 pathway reduced sphere expansion. The enhanced self-renewal activity of the CXCR4-positive spheres was preceded by the upregulation of HIF2α. Knockdown of HIF2α abrogated CXCR4 expression and sphere formation. Finally, RCC-derived spheres showed an undifferentiated phenotype in vivo and formed subcutaneous tumours that highly expressed HIF2α and CXCR4. Inhibition of HIF2α abolished tumour growth in animal models.

Conclusions:

These results suggest that the generation of RCC-derived CSCs involves the activation of HIF2α and may provide a foundation for the development of new strategies to prevent the induction of CSCs in RCC.     

CD8+ T cells undergo activation and programmed death-1 repression in the liver of aged Ae2a,b?/? mice favoring autoimmune cholangitis

Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl⁻/HCO₃⁻ anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2_a,b^−/− mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2_a,b^−/− mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8⁺ T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2′-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8⁺ T cells from aged knockouts. Conclusion: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8⁺ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.     

Functional CD3+CD8+PD1− T Cell Accumulation and PD-L1 Expression Increases During Tumor Invasion in DCIS of the Breast

BackgroundThe changes in T cell subsets and programmed death ligand 1 (PD-L1) expression during the transition from ductal carcinoma in situ (DCIS) to early invasive breast cancer had not been well studied.Patients and MethodsA total of 85 DCIS patients were classified into 49 DCIS (clinical stage: Tis, noninvasive) and 36 with a minimally infiltrating lesion (MIL; < 5 mm; clinical stage: T1a). We explored the quantitative alterations of T-cell markers and PD-L1 in these groups using the Opal multi-immunohistochemistry technique.ResultsWe observed increased infiltration of CD3-positive (CD3⁺)CD8⁺ programmed death 1 (PD1)-negative T cells and higher PD-L1 expression in DCIS with MIL. Elevated PD1 expression correlated with PD-L1 expression in MIL and DCIS.ConclusionWe conclude that during the transition from DCIS to an invasive lesion, the host cytolytic T cells begin interacting with the tumor and destroy the tumor tissue, leading to an adaptive upregulation of PD-L1 and tumor protection against immune destruction.     

Upregulation of CD9 in ovarian cancer is related to the induction of TNF-α gene expression and constitutive NF-κB activation

Ovarian cancer is a gynecological cancer with a high death rate. We utilized global gene expression profiles of ovarian carcinomas obtained by complementary DNA (cDNA) microarray to identify ovarian cancer-specific proteins. CD9 was upregulated in ovarian carcinomas, and overexpression of the CD9 protein was detected in ovarian carcinomas by immunohistochemistry. CD9 was also overexpressed in several cancer cell lines, including ovarian cancer cells. In order to elucidate the biological significance of highly expressed CD9 in cancer cells, functional studies of CD9 were performed by ectopic expression, knockdown of CD9 using small interfering RNA (siRNA) and blockage of CD9 activity using the CD9-specific monoclonal antibody ALB6. Ectopic CD9 induced cell survival. In order to identify signaling pathways related to CD9, the gene expressions of CD9/SKOV3 cells were analyzed by cDNA microarray. Among the many upregulated genes, tumor necrosis factor (TNF)-α was induced in CD9/SKOV3 cells. The effect of overexpressed CD9 on the downstream signaling events of TNF-α was further investigated. In CD9/SKOV3 cells, the nuclear factor-kappaB (NF-κB)-signaling pathway was constitutively activated. Knockdown of CD9 by siRNA and blockage of CD9 activity by ALB6 in ovarian cancer cells demonstrated that constitutive activation of NF-κB is CD9 dependent and that CD9 is involved in anti-apoptosis. A CD9 functional study was performed in an ovarian cancer-xenograft mouse by injecting ALB6 into the peritoneum. ALB6 resulted in reduced tumor weight compared with that of control IgG(1). Collectively, these results demonstrate that CD9 functions as an oncogene and represents a target for the development of cancer-specific therapeutics.