Induction Of Immunosuppressive Cells Research Articles

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression

ABSTRACT The tumor microenvironment (TME) in ovarian cancer (OC) is characterized by immune suppression, due to an abundance of suppressive immune cells populations. To effectively enhance the activity of immune checkpoint inhibition (ICI), there is a need to identify agents that target these immunosuppressive networks while promoting the recruitment of effector T cells into the TME. To this end, we sought to investigate the effect of the immunomodulatory cytokine IL12 alone or in combination with dual-ICI (anti-PD1 + anti-CTLA4) on anti-tumor activity and survival, using the immunocompetent ID8-VEGF murine OC model. Detailed immunophenotyping of peripheral blood, ascites, and tumors revealed that durable treatment responses were associated with reversal of myeloid cell-induced immune suppression, which resulted in enhanced anti-tumor activity by T cells. Single cell transcriptomic analysis further demonstrated striking differences in the phenotype of myeloid cells from mice treated with IL12 in combination with dual-ICI. We also identified marked differences in treated mice that were in remission compared to those whose tumors progressed, further confirming a pivotal role for the modulation of myeloid cell function to allow for response to immunotherapy. These findings provide the scientific basis for the combination of IL12 and ICI to improve clinical response in OC.

Drug-free biodegradable nanoparticles alleviate myeloid cell-induced immunosuppression and inhibit metastasis.

e14555 Background: Metastasis is responsible for most cancer-related deaths and depends on the formation of a metastatic niche at distal sites. The metastatic niche consists primarily of immunosuppressive myeloid cells, such as neutrophils and monocytes. These cells are recruited to distal sites in response to cancer-induced immune dysregulation and promote the recruitment and survival of disseminated tumor cells. We have employed drug-free poly(lactide-co-glycolide) (PLGA) nanoparticles, ONP-302, to modulate myeloid cell phenotypes and trafficking to disrupt the formation of the metastatic niche. The anti-metastatic efficacy of ONP-302 nanoparticles was evaluated in a translationally relevant mouse model of orthotopic triple-negative breast cancer. Methods: ONP-302 nanoparticles were evaluated in the syngeneic 4T1 model of triple-negative breast cancer in female BALB/c or RAG1KO BALB/c mice. ONP-302 nanoparticles intravenously injected into tumor-bearing mice every 3 days starting 1 day after tumor inoculation. Lungs were extracted 14 and 21 days after inoculation for analysis by single cell RNA sequencing and quantification of metastases by luminescent imaging, respectively. In resection studies, primary tumors were resected 11 days after inoculation and tissues were extracted 42 days after resection for quantification of metastases. Results: ONP-302 nanoparticles significantly reduced primary tumor growth and completely inhibited the formation of lung metastases. ONP-302 was also effective at suppressing metastasis when applied following surgical resection. In mice which received ONP-302 after surgical resection, lung metastases were undetectable up to 42 days after resection, while 44% of control mice developed metastases. These findings suggest that ONP-302 targets both primary tumor growth and metastatic colonization. Single cell RNA sequencing was employed to analyze ONP-302 modulation of the lung metastatic niche. The lungs of ONP-302 treated mice had lower neutrophil infiltration and a higher proportion of monocytes and dendritic cells compared to control mice. Gene expression pathways associated with inflammatory interferon and tumor necrosis factor responses were upregulated in neutrophils, monocytes, and dendritic cells from ONP-302 treated mice. The upregulation of these pathways suggests that ONP-302 treatment prevents the formation of an immunosuppressive niche and promotes anti-tumor cytotoxicity. Adaptive immunity was essential for ONP-302 efficacy, as T and B cell deficient RAG1KO mice demonstrated complete loss of ONP-302 efficacy both in slowing primary tumor growth and preventing metastasis. Conclusions: Our findings demonstrate that our drug-free PLG nanoparticles, ONP-302, impede cancer metastasis as a monotherapy by inhibiting the accumulation of immunosuppressive myeloid cells.

Idelalisib Rescues Natural Killer Cells from Monocyte-Induced Immunosuppression by Inhibiting NOX2-Derived Reactive Oxygen Species.

The phosphatidylinositol-4,5-bisphosphate-3 kinase-δ (PI3Kδ) inhibitor idelalisib, used alone or in combination with anti-CD20, is clinically efficacious in B-cell lymphoma and chronic lymphocytic leukemia (CLL) by promoting apoptosis of malignant B cells. PI3K regulates the formation of reactive oxygen species (ROS) by the myeloid NADPH oxidase NOX2, but the role of PI3Kδ in myeloid cell-induced immunosuppression is unexplored. We assessed the effects of idelalisib on the spontaneous and IgG antibody-induced ROS production by human monocytes, on ROS-induced cell death of human natural killer (NK) cells, and on tumor cell clearance in an NK cell-dependent mouse model of metastasis. Idelalisib potently and efficiently inhibited the formation of NOX2-derived ROS from monocytes and rescued NK cells from ROS-induced cell death. Idelalisib also promoted NK cell cytotoxicity against anti-CD20-coated primary human CLL cells and cultured malignant B cells. Experiments using multiple PI3K inhibitors implicated the PI3Kδ isoform in regulating NOX2-induced ROS formation and immunosuppression. In B6 mice, systemic treatment with idelalisib significantly reduced the formation of lung metastases from intravenously injected melanoma cells but did not affect metastasis in B6.129S6-Cybbtm1Din (Nox2 -/-) mice or in NK cell-deficient mice. Our results imply that idelalisib rescues NK cells from NOX2/ROS-dependent immunosuppression and thus exerts antineoplastic efficacy beyond B-cell inhibition.

Targeting Myeloid-Derived Suppressor Cells in Cancer Immunotherapy.

Myeloid-derived suppressor cells (MDSCs), which are activated under pathological conditions, are a group of heterogeneous immature myeloid cells. MDSCs have potent capacities to support tumor growth via inhibition of the antitumoral immune response and/or the induction of immunosuppressive cells. In addition, multiple studies have demonstrated that MDSCs provide potential therapeutic targets for the elimination of immunosuppressive functions and the inhibition of tumor growth. The combination of targeting MDSCs and other therapeutic approaches has also demonstrated powerful antitumor effects. In this review, we summarize the characteristics of MDSCs in the tumor microenvironment (TME) and current strategies of cancer treatment by targeting MDSCs.

IMMU-36. THE ROLE OF PD-L1 IN GLIOBLASTOMA-DERIVED EXTRACELLULAR VESICLES IN THE INDUCTION OF IMMUNOSUPPRESSIVE MONOCYTES

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor, and novel therapeutic strategies that make a substantial impact on outcomes are sorely needed. Immunotherapies have shown great promise in the treatment of a number of cancers in recent year, and a concerted effort is being made to apply these treatment paradigms to GBM. However, many GBM patients exhibit profound immunosuppression, likely limiting the efficacy of such approaches. The mechanisms of this immunosuppression are poorly understood, but tumor-derived extracellular vesicles (EVs), may play a role. We demonstrate that GBM-derived EVs induce the development of myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs). We further demonstrate that these EV-induced monocytic cells are immunosuppressive, resulting in impaired T cell proliferation upon co-culture. We found that the immunosuppressive effects of tumor-derived EVs appear to be driven by the induction of these immunosuppressive monocytes, as EV treatment of T cells did not significantly impact T cell proliferation. Further, we sought to characterize the important of programmed death ligand 1 (PD-L1) in the induction of these immunosuppressive monocyte populations. We found PD-L1 to be expressed in the EVs from GBM cell lines, and that modulation in PD-L1 expression via either constitutive overexpression or shRNA-mediated knockdown resulted in concordant changes in expression in tumor-derived EVs. We demonstrate that PD-L1 is important for the induction of NCM but not for MDSCs. Taken together, these findings point to a significant role for tumor-derived EVs in the induction of immunosuppressive monocytes in GBM, and that these cells may be a driving force of systemic immunosuppression. PD-L1 is one factor expressed in EVs that has immunomodulatory properties, but additional EV cargo likely plays a major role in the induction of immunosuppressive cells.

Biocompatibility of HbV: Liposome-Encapsulated Hemoglobin Molecules-Liposome Effects on Immune Function

Hemoglobin vesicles (HbVs) are oxygen carriers consisting of Hb molecules and liposome in which human hemoglobin (Hb) molecules are encapsulated. Investigations of HbV biocompatibility have shown that HbVs have no significant effect on either the quality or quantity of blood components such as RBC, WBC, platelets, complements, or coagulation factors, reflecting its excellent biocompatibility. However, their effects on the immune system remain to be evaluated. HbVs might affect the function of macrophages because they accumulate in the reticuloendothelial system. Results show that splenic T cell proliferation is suppressed after injection of not only HbV but also empty liposome into rat, and show that macrophages that internalized liposomal particles are responsible for the suppression. However, the effect is transient. Antibody production is entirely unaffected. Further investigation revealed that those macrophages were similar to myeloid-derived suppressor cells (MDSCs) in terms of morphology, cell surface markers, and the immune-suppression mechanism. Considering that MDSCs appear in various pathological conditions, the appearance of MDSC-like cells might reflect the physiological immune system response against the substantial burden of liposomal microparticles. Therefore, despite the possible induction of immunosuppressive cells, HbVs are an acceptable and promising candidate for use as a blood substitute in a clinical setting.

OS09.2 Enhancing immunity while neutralizing T cell-induced immunosuppression through combinatorial immunotherapy of glioblastoma

OS09.2 Enhancing immunity while neutralizing T cell-induced immunosuppression through combinatorial immunotherapy of glioblastoma

Old Mice Accumulate Activated Effector CD4 T Cells Refractory to Regulatory T Cell-Induced Immunosuppression.

Chronic low-grade inflammation and reduced lymphocyte potency are implicated in the pathogenesis of major illnesses associated with aging. Whether this immune phenotype results from a loss of cell-mediated regulation or intrinsic dysregulated function of effector T cells (Teffs) requires further research. Here, we report that, as compared with young C57BL6 mice, old mice show an increased frequency of CD4+CD62L− Teffs with a dysregulated activated phenotype and markedly increased effector functions. Analysis of the frequency and suppressive function of CD4+FoxP3+ regulatory T cells (Tregs) indicates an increase in the frequency of FoxP3+ T cells with aging which, however, occurs within the CD4+CD25− T cells. Furthermore, whereas Tregs from young and old mice similarly suppress Teffs from young mice, both have a compromised suppressive capacity of Teffs from old mice, a phenomenon which is partially recovered in the presence of IL-2-producing CD4+CD62L+ non-Teffs. Finally, we observed that Teff subsets from old mice are enriched with IL-17A-producing T cells and exhibit intrinsically dysregulated expression of genes encoding cell-surface molecules and transcription factors, which play a key role in T-cell activation and regulation. We, thus, demonstrate an age-related impairment in the regulation of effector CD4 T cells, which may underlie the higher risk for destructive inflammation associated with aging.

The immunobiology of myeloid-derived suppressor cells in cancer.

The tumor microenvironment is a complex and heterogeneous milieu in which multiple interactions occur between tumor and host cells. Immunosuppressive cells which are present in this microenvironment, such as regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs), play an important role in tumor progression, via down-regulation of antitumor responses. MDSCs represent a heterogeneous group of cells originated from the myeloid lineage that are in the immature state. These cells markedly accumulate under pathologic conditions, such as cancer, infection, and inflammation, and use various mechanisms to inhibit both adaptive and innate immune responses. These immunosuppressive mechanisms include deprivation of T cells from essential amino acids, induction of oxidative stress, interference with viability and trafficking of T cells, induction of immunosuppressive cells, and finally polarizing immunity toward a tumor-promoting type 2 phenotype. In addition to suppression of antitumor immune responses, MDSCs can also enhance the tumor metastasis and angiogenesis. Previous studies have shown that increased frequency of MDSCs is related to the tumor progression. Moreover, various drugs that directly target these cells or reverse their suppressive activity can improve antitumor immune responses as well as increase the efficacy of immunotherapeutic intervention. In this review, we will first discuss on the immunobiology of MDSCs in an attempt to find the role of these cells in tumor progression and then discuss about therapeutic approaches to target these cells.

Immunosuppression associated with chronic inflammation in the tumor microenvironment.

Chronic inflammation contributes to cancer development via multiple mechanisms. One potential mechanism is that chronic inflammation can generate an immunosuppressive microenvironment that allows advantages for tumor formation and progression. The immunosuppressive environment in certain chronic inflammatory diseases and solid cancers is characterized by accumulation of proinflammatory mediators, infiltration of immune suppressor cells and activation of immune checkpoint pathways in effector T cells. In this review, we highlight recent advances in our understanding of how immunosuppression contributes to cancer and how proinflammatory mediators induce the immunosuppressive microenvironment via induction of immunosuppressive cells and activation of immune checkpoint pathways.

Enhanced anti-tumor effects of the PD-1/PD-L1 blockade by combining a highly absorptive form of NF-kB/STAT3 inhibitor curcumin

The PD-1/PD-L1 blockade is now recognized as one of the basic immune interventions for development of combination cancer immunotherapy. We have been screening chemical libraries to obtain compounds which have an activity to augment anti-tumor activity of the PD-1/PD-L1 blockade. We have previously shown that activation of NF-kB and STAT3 signals in both cancer cells and immune cells plays important roles in cancer cell-induced immunosuppression, and their inhibition may augment anti-tumor CTL. Curcumin, a major active component of turmeric, has activities to inhibit both NF-kB and STAT3 signals. We have recently developed a highly absorptive form of curcumin which oral administration resulted in high plasma concentration. In this study, we evaluated combination treatment of this highly absorptive form of curcumin with the PD-1/PD-L1 blockade. Curcumin inhibited significantly in vitro production of IL6 and IL8 by human ovarian cancer cell lines (OC) having activated NF-kB and STAT3 signaling. In nude mice implanted with human OC, T-cell stimulatory activity of murine DC was impaired partly through increased human IL-6 from human OC. Systemic p.o. administration of curcumin decreased human IL6 in mouse serum and restored T-cell stimulatory activity of murine DC in spleens and tumors. In two syngeneic murine colon cancer models, NF-kB dependent IL6 producing MC38 in B6 mice and NF-kB non-activated IL6 non-producing CT26 in Balb/C mice, curcumin p.o. administration resulted in the significant augmentation of tumor antigen-specific CD8+ T-cell induction accompanied by increased T-cell stimulatory activity of DC via decreased STAT3 and NF-kB signaling, although curcumin did not inhibit proliferation of both tumor cell lines. Furthermore, p.o. administration of curcumin into non-tumor bearing B6 mice immunized with ovalbumin (OVA) peptide augmented OVA-specific CTL induction. These results indicate that curcumin not only enhances tumor antigen specific T-cell via reversal of tumor-induced immunosuppression (e.g. IL6 induced DC impairment), but also enhances CTL via directly acting on immune cells. Since PD-1 positive T cells were infiltrated in tumors expressing PD-L1, we evaluated combination of curcumin and anti-PD-L1 blockade, and found that combination of curcumin and anti-PD-L1 Ab had synergistic anti-tumor activity. These results indicate that combination of curcumin which enhances induction of tumor antigen specific, PD-1 positive CTL in tumors via acting on both cancer cells and immune cells and local PD-1/PD-L1 blockade, is an attractive strategy for development of effective cancer immunotherapy.

Control of the immune response by pro-angiogenic factors.

The progressive conversion of normal cells into cancer cells is characterized by the acquisition of eight hallmarks. Among these criteria, the capability of the cancer cell to avoid the immune destruction has been noted. Thus, tumors develop mechanisms to become invisible to the immune system, such as the induction of immunosuppressive cells, which are able to inhibit the development of an efficient immune response. Molecules produced in the tumor microenvironment are involved in the occurrence of an immunosuppressive microenvironment. Recently, it has been shown that vascular endothelial growth factor A (VEGF-A) exhibits immunosuppressive properties in addition to its pro-angiogenic activities. VEGF-A can induce the accumulation of immature dendritic cells, myeloid-derived suppressor cells, regulatory T cells, and inhibit the migration of T lymphocytes to the tumor. Other pro-angiogenic factors such as placental growth factor (PlGF) could also participate in tumor-induced immunosuppression, but only few works have been performed on this point. Here, we review the impact of pro-angiogenic factors (especially VEGF-A) on immune cells. Anti-angiogenic molecules, which target VEGF-A/VEGFR axis, have been developed in the last decades and are commonly used to treat cancer patients. These drugs have anti-angiogenic properties but can also counteract the tumor-induced immunosuppression. Based on these immunomodulatory properties, anti-angiogenic molecules could be efficiently associated with immunotherapeutic strategies in preclinical models. These combinations are currently under investigation in cancer patients.

Role of OGR1 in myeloid-derived cells in prostate cancer

Ovarian cancer G-protein-coupled-receptor-1 (OGR1) is a tumor metastasis suppressor in prostate cancer (PCa). OGR1 knockout mice (ogr1(-/-)) are grossly normal under physiological conditions, however, reduced melanoma tumorigenesis has been observed, with the mechanisms of this reduction completely unknown. In this work, we demonstrated that OGR1 deficiency in host cells significantly reduced tumorigenesis of PCa in mice. Adoptive transfer of WT CD11b(+) Gr1(+) double positive (DP) cells, but not T cells, was sufficient to allow tumor development in ogr1(-/-) mice. The expression of an M1 macrophage marker, inducible nitric oxide synthase (iNOS) was higher and expression of an M2 macrophage marker, arginase-1 (Arg 1) was lower in tumors from ogr1(-/-) mice compared with WT mice. Furthermore, coinjection of transgenic adenocarcinoma mouse prostate (TRAMP)-C2 cells with WT, but not ogr1(-/-) macrophages, increased tumor incidence in ogr1(-/-) mice. T-cell depletion experiments suggested that T cells were required for tumor rejection in ogr1(-/-)mice, although OGR1 expression in T cells may not be necessary. In summary, the expression of OGR1 in myeloid-derived cells, especially in DP cells, was required for PCa tumor cell-induced immunosuppression.

Comparison of Foxp3+ Regulatory T cells and CD163+ Macrophages in Invasive and Non-invasive Extramammary Paget’s Disease

Regulatory T cells (Tregs), identified by the expression of CD4, CD25 and Foxp3, together with immunosuppressive macrophages, such as CD163+ M2 macrophages, are involved in maintaining peripheral tolerance. The aim of this study was to elucidate the involvement of Tregs and CD163+ macrophages in invasive and non-invasive extramammary Paget's disease. The presence of CD4+CD25+Foxp3+ Tregs, CD163+ M2 macrophages and matrix metalloproteinase-9+ cells was examined immunohistologically in fixed sections of lesional skin from 10 patients with non-invasive extramammary Paget's disease and 7 patients with invasive extramammary Paget's disease. Fewer CD4+CD25+Foxp3+ Tregs were observed in non-invasive extramammary Paget's disease than in invasive extramammary Paget's disease. In contrast, higher numbers of CD163+ macrophages and metalloproteinase-9+ cells were detected only in invasive extramammary Paget's disease. These findings suggest that the induction of immunosuppressive cells in extramammary Paget's disease differs according to the tumour stage.

Systemic Immune Tuning in Renal Cell Carcinoma

Renal cell carcinoma (RCC) can inhibit protective immunity by induction of immunosuppressive cells that produce inhibitory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β. If this immunosuppression influences response to kinase inhibitors such as sorafenib is not known. Therefore, we asked for the prognostic influence of cells with immunosuppressive properties in peripheral blood (pB) in a cohort of metastatic clear cell renal cell carcinoma (mRCC) patients uniformly receiving sorafenib treatment. IL-10 and TGF-β mRNA levels, regulatory T-cell (Treg) counts, and frequencies of IL-10/TGF-β producing mononuclear cell subsets were determined in pB from 46 patients with mRCC before receiving sorafenib treatment. Relationship between established clinical and laboratory prognostic factors and outcome were examined by univariate and multivariate Cox regression analysis. IL-10 and TGF-β1 mRNA levels, and frequencies of CD4(+)CD25high/CD3(+) and CD4(+)CD25highFoxP3(+)/CD3(+)Treg cells were significantly higher in mRCC patients compared with healthy individuals. Monocytes were suggested as main producers of IL-10 and TGF-β. In a multivariate analysis low ECOG score and-surprisingly-high TGF-β1 mRNA levels were independently associated with favorable progression-free survival (P=0.005 and P=0.003, respectively) and overall survival (P=0.001 and P=0.039, respectively). In conclusion, mRCC is associated with an immunosuppressive phenotype in peripheral blood. The positive prognostic influence of high TGF-β1 mRNA expression levels may reflect immune promoting functions of TGF-β in combined activity with inflammatory cytokines.

Anti–Transforming Growth Factor β Receptor II Antibody Has Therapeutic Efficacy against Primary Tumor Growth and Metastasis through Multieffects on Cancer, Stroma, and Immune Cells

Transforming growth factor beta (TGFbeta) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFbeta receptor II (TGFbeta RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models. We generated neutralizing antibodies to TGFbeta RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models. Anti-TGFbeta RII antibody blocked TGFbeta RII binding to TGFbeta 1, 2, and 3, and attenuated the TGFbeta-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumor-bearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis. These preclinical data provide a foundation to support using anti-TGFbeta RII antibody as a therapeutic agent for TGFbeta RII-dependent cancer with metastatic capacity.

Immune Self-tuning in Renal Cell Carcinoma

Although renal cell carcinoma (RCC) is an immunogenic tumour, and although there is evidence that in a small proportion of cases antitumour immune responses may mediate tumour regression or at least disease stabilisation, patients with progressive disease have no effective antitumour immune response. Besides preventing recognition of the tumour by immune effector cells, RCC escapes the immune system by induction of tolerance through manipulating the function and proliferation of immune effector cells. This tuning of the immune response can occur by active suppression of immune effector cells through inhibitory molecules expressed on the tumour surface and through various tumour-secreted soluble factors, or it can be mediated indirectly by induction of immunosuppressive cells. This review provides an overview of the most common mechanisms that mediate immune tolerance in RCC and discusses the therapeutic perspectives of immunoregulatory strategies in the era of targeted therapies.

Role of Invariant Natural Killer T Cells in Liver Sinusoidal Endothelial Cell-Induced Immunosuppression Among T Cells with Indirect Allospecificity

We have previously demonstrated that liver sinusoidal endothelial cells (LSECs) that endocytose portally injected allogeneic cells exert immunosuppressive effects on T cells with indirect allospecificity. In this study, we have demonstrated that invariant natural killer T cells plays a significant role in such immunosuppressive effects induced by LSECs. The endocytic activity of LSECs toward intraportally injected splenocytes from B6 major histocompatibility complex class II-deficient C2ta (C2D) mice was markedly impaired in BALB/c CD1d-deficient mice. The intraportal adoptive transfer of LSECs isolated from BALB/c wild-type mice treated with a portal injection of B6 C2D splenocytes into BALB/c mice significantly prolonged the survival of subsequently transplanted heart allografts; however, the transfer of LSECs isolated from similarly treated BALB/c CD1d-deficient mice did not produce such a survival prolonging effect. These findings indicate that natural killer T cells are required for the LSEC-induced immune modulation of T cells with indirect allospecificity.

High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impair the immune response through the recruitment of myeloid suppressor cells.

Tumor vaccines have shown promise in early clinical trials. Among them, tumor cells genetically engineered to secrete biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF) can generate a systemic antitumor immune response. Although the minimal required GM-CSF dose produced by modified tumor cells to achieve a measurable antitumor effect is well known, no data examined whether an upper therapeutic limit may exist for this vaccination strategy. Because recent data demonstrate an immunosuppressive effect of GM-CSF produced by growing tumors, we thus sought to determine whether high GM-CSF doses administered in a vaccine formulation could impair antitumor immunity. Using a vaccine strategy involving a GM-CSF-producing bystander cell line (B78H1-GM) admixed with autologous tumor, we assessed the impact of varying doses of GM-CSF while maintaining a constant antigen dose. Our results defined a threshold above which a GM-CSF-based vaccine not only lost its efficacy, but more importantly for its clinical implications resulted in substantial immunosuppression in vivo. Above this threshold, GM-CSF induced Gr1+/CD11b+ myeloid suppressor cells that substantially impaired antigen-specific T-cell responses and adversely affected antitumor immune responses in vivo. The dual effects of GM-CSF are mediated by the systemic and not local concentration of this cytokine. Myeloid suppressor cell-induced immunosuppression is mediated by nitric oxide production via inducible nitric oxide synthase (iNOS) because the specific iNOS inhibitor, l-NMMA, restored antigen-specific T-cell responsiveness in vitro. Taken together, our data demonstrated the negative impact of supra-therapeutic vaccine doses of GM-CSF and underscored the importance of identifying these critical variables in an effort to increase the therapeutic efficacy of tumor vaccines.