Tumor Microenvironment CD14+ Cells Correlate with Poor Overall Survival in Patients with Early-Stage Lung Adenocarcinoma.

Patients with early-stage lung adenocarcinoma have a high risk of recurrent or metastatic disease despite undergoing curative intent therapy. We hypothesized that increased CD14+ cells within the tumor microenvironment (TME) could stratify patient outcomes. Immunohistochemistry for CD14 was performed on 189 specimens from patients with lung adenocarcinoma who underwent curative intent surgery. Outcomes and associations with clinical and pathologic variables were determined. In vitro studies utilized a coculture system to model the lung cancer TME containing CD14+ cells. Patients with high levels of TME CD14+ cells experienced a median overall survival of 5.5 years compared with 8.3 and 10.7 years for those with moderate or low CD14 levels, respectively (p < 0.001). Increased CD14+ cell tumor infiltration was associated with a higher stage at diagnosis and more positive lymph nodes at the time of surgery. This prognostic capacity remained even for patients with early-stage disease. Using an in vitro model system, we found that CD14+ cells reduced chemotherapy-induced cancer cell death. These data suggest that CD14+ cells are a biomarker for poor prognosis in early-stage lung adenocarcinoma and may promote tumor survival. CD14+ cell integration into the lung cancer TME can occur early in the disease and may be a promising new therapeutic avenue.

Novel strategy for manufacturing autologous dendritic cell/allogeneic tumor lysate vaccines for glioblastoma.

BACKGROUND: Glioblastoma, the most common primary malignant brain tumor, is nearly universally fatal by 5 years. Dendritic cell vaccines are promising but often limited clinically by antigen choice, dendritic cell potency, and/or manufacturing yield. We optimized vaccine manufacture, generating potent mature autologous dendritic cells pulsed with allogeneic glioblastoma lysates. METHODS: Platelet lysate-based supplement was used to establish human glioblastoma cell lines. Phenotype and genotype were assessed. An improved culture technique to generate mature dendritic cells from glioblastoma patients' monocytes was developed. The ability of T cells stimulated with autologous dendritic cells pulsed with allogeneic glioblastoma cell lysate to kill HLA-A2-matched glioblastoma cells was assessed. RESULTS: Glioblastoma cell lines established with platelet lysate supplement grew faster and expressed more stem-like markers than lines grown in neural stem cell media or in the presence of serum. They expressed a variety of glioma-associated antigens and had genomic abnormalities characteristic of glioblastoma stable up to 15 doublings. Unlike standard culture techniques, our optimized technique produced high levels of mature dendritic cells from glioblastoma patients' monocytes. Autologous T cells stimulated with mature dendritic cells pulsed with allogeneic glioblastoma cell line lysate briskly killed HLA-A2-matched glioblastoma cells. CONCLUSIONS: Our glioblastoma culture method provides a renewable source for a broad spectrum glioblastoma neoantigens while our dendritic cell culture technique results in more mature dendritic cells in glioblastoma patients than standard techniques. This broadly applicable strategy could be easily integrated into patient care.

Mesenchymal stromal cells protect human cardiomyocytes from amyloid fibril damage.

BACKGROUND AIMS: Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils. METHODS: We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils. RESULTS: We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease. CONCLUSIONS: This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.

Immune independent crosstalk between lymphoma and myeloid suppressor CD14+HLA-DRlow/neg monocytes mediates chemotherapy resistance.

We have previously reported a novel phenotype of myeloid suppressors in lymphoma patients characterized by a loss of HLA-DR expression on monocytes, CD14+HLA-DRlow/neg. These cells were directly immunosuppressive and were associated with poor clinical outcome. In this study, we found that lymphoma tumors could have more than 30% of their tumor occupied by CD14+ cells. This intimate spatial connection suggested substantial cell-cell communication. We examined cross talk between monocytes from healthy volunteers (normal) and lymphoma cells in co-culture to identify the mechanisms and consequences of these interactions. Normal CD14+HLA-DR+ monocytes lost their HLA-DR expression after co-culture with lymphoma cells. Lymphoma-converted CD14+HLA-DRlow/neg cells exhibited similar immunosuppressive functions as CD14+HLA-DRlow/neg monocytes from lymphoma patients. Unexpectedly monocyte additions to lymphoma cell cultures protected lymphoma from cytotoxic killing by chemotherapy drug doxorubicin (DOX). Monocyte mediated resistance to DOX killing was associated with decreased Caspase-3 activity and increased anti-apoptotic heat shock protein-27 (Hsp27) expression. Soluble Hsp27 was detected in supernatant and patient plasma. Increased Hsp27 in plasma correlated with increased proportion of CD14+HLA-DRlow/neg monocytes in patient blood and was associated with lack of clinical response to DOX. This is the first report to describe a non-immune function of CD14+HLA-DRlow/neg monocytes: enhanced lymphoma resistance to chemotherapy. It is also the first report in lymphoma of Hsp27 as a potential mediator of lymphoma and monocyte crosstalk and chemotherapy resistance. Together with previous reports of the prevalence of these myeloid suppressors in other cancers, our findings identify this pathway and these interactions as a potential novel therapeutic target.

Intratumoral CD14+ Cells and Circulating CD14+HLA-DRlo/neg Monocytes Correlate with Decreased Survival in Patients with Clear Cell Renal Cell Carcinoma.

PURPOSE: Immunotherapeutic strategies to treat patients with renal cell carcinoma (RCC) offer new opportunities for disease management. Further improvements to immunotherapy will require additional understanding of the host response to RCC development. EXPERIMENTAL DESIGN: Using a novel approach to understanding the immune status of cancer patients, we previously showed that patients with a certain immune profile had decreased overall survival. Here, we examine in more detail the phenotypic changes in peripheral blood and the potential consequences of these changes in RCC patients. RESULTS: We found that CD14(+)HLA-DR(lo/neg) monocytes were the most predominant phenotypic change in peripheral blood of RCC patients, elevated nearly 5-fold above the average levels measured in healthy volunteers. Intratumoral and peritumoral presence of CD14 cells was an independent prognostic factor for decreased survival in a cohort of 375 RCC patients. The amount of peripheral blood CD14(+)HLA-DR(lo/neg) monocytes was found to correlate with the intensity of CD14 staining in tumors, suggesting that the measurement of these cells in blood may be a suitable surrogate for monitoring patient prognosis. The interaction of monocytes and tumor cells triggers changes in both cell types with a loss of HLA-DR expression in monocytes, increases of monocyte survival factors such as GM-CSF in tumors, and increased production of angiogenic factors, including FGF2. CONCLUSIONS: Our results suggest a model of mutually beneficial interactions between tumor cells and monocytes that adversely affect patient outcome.

Cancer Vaccines in the World of Immune Suppressive Monocytes (CD14(+)HLA-DR(lo/neg) Cells): The Gateway to Improved Responses.

Dendritic cells are an important target in cancer immunotherapy based on their critical role in antigen presentation and response to tumor development. The capacity of dendritic cells to stimulate anti-tumor immunity has led investigators to use these cells to mediate anti-tumor responses in a number of clinical trials. However, these trials have had mixed results. The typical method for generation of ex vivo dendritic cells starts with the purification of CD14(+) cells. Our studies identified a deficiency in the ability to generate mature dendritic cell using CD14(+) cells from cancer patients that corresponded with an increased population of monocytes with altered surface marker expression (CD14(+)HLA-DR(lo/neg)). Further studies identified systemic immune suppression and increased concentrations of CD14(+)HLA-DR(lo/neg) monocytes capable of inhibiting T-cell proliferation and DC maturation. Together, these findings strongly suggest that protocols aimed at immune stimulation via monocytes/dendritic cells, if optimized on normal monocytes or in systems without these suppressive monocytes, are unlikely to engender effective DC maturation in vitro or efficiently trigger DC maturation in vivo. This highlights the importance of developing optimal protocols for stimulating DCs in the context of significantly altered monocyte phenotypes often seen in cancer patients.

Soluble B7-H1: differences in production between dendritic cells and T cells.

Tumor cells aberrantly express several T cell inhibitory molecules including members of the B7-H co-regulatory family. Presumably tumor-expressed B7-H1 and B7-H3 confer resistance to elimination by the immune system. In addition, elevated levels of soluble B7-H1 (sB7-H1) has been identified in the sera of cancer patients, including renal carcinoma patients and is associated with increased cancer related death. Here we report that sB7-H1 is produced and released by activated mature dendritic cells (mDC). Immature DC, macrophages, monocytes, or T cells are refractory to releasing sB7-H1. Exposure of CD4+ and CD8+ T cells to mDC-derived sB7-H1 molecules induced apoptosis. These data suggest that the immunobiology of B7-H1 is perhaps more complex than previously thought. sB7-H1 molecules may represent an unanticipated contributing factor to immune homeostasis. That both immune and tumor cells can be sources of sB7-H1 suggests that optimization of co-regulatory blockade immunotherapy for solid malignancies of necessity will require impact of targeting tumor and immune-derived B7-H1 molecules.

Immunosuppressive CD14+HLA-DR(low)/- monocytes in B-cell non-Hodgkin lymphoma.

Immunosuppression is a known risk factor for B-cell non-Hodgkin lymphoma (NHL), yet mechanisms of tumor-associated immunosuppression remain to be fully characterized. We examined the immunophenotype of 40 NHL patients and 27 age-matched healthy volunteers to better understand systemic immune suppression. NHL peripheral blood mononuclear cells had significantly decreased interferon-γ production and proliferation. This suppression was not the result of regulatory T cells, interleukin-6 or interleukin-10, as these factors were not different between NHL and healthy volunteers (controls). We were able to restore T-cell proliferation by removing NHL monocytes, suggesting that these monocytes are suppressive. This suppression was mediated in part through arginine metabolism as exogenous arginine supplementation partially overcame monocytes' suppression of T-cell proliferation in vitro and NHL patients had elevated arginase I in their plasma. NHL monocytes had impaired STAT1 phosphorylation and interferon-α production to CpG stimulation and a dendritic cell differentiation deficiency. Further studies demonstrated that monocytes from NHL patients had decreased HLA-DR and Tumor necrosis factor-α receptor II (CD120b) expression compared with controls (CD14(+)HLA-DR(low/-)CD120b(low)). Patients with increased ratios of CD14(+)HLA-DR(low/-) monocytes had more aggressive disease and suppressed immune functions. In summary, we report that CD14(+)HLA-DR(low/-) monocytes are a major and multifactorial contributor to systemic immunosuppression in NHL.

Systemic immune suppression in glioblastoma: the interplay between CD14+HLA-DRlo/neg monocytes, tumor factors, and dexamethasone.

Patients with glioblastoma (GBM) exhibit profound systemic immune defects that affect the success of conventional and immune-based treatments. A better understanding of the contribution of the tumor and/or therapy on systemic immune suppression is necessary for improved therapies, to monitor negative effects of novel treatments, to improve patient outcomes, and to increase understanding of this complex system. To characterize the immune profile of GBM patients, we phenotyped peripheral blood and compared these to normal donors. In doing so, we identified changes in systemic immunity associated with both the tumor and dexamethasone treated tumor bearing patients. In particular, dexamethasone exacerbated tumor associated lymphopenia primarily in the T cell compartment. We have also identified unique tumor and dexamethasone dependent altered monocyte phenotypes. The major population of altered monocytes (CD14(+)HLA-DR(lo/neg)) had a phenotype distinct from classical myeloid suppressor cells. These cells inhibited T cell proliferation, were unable to fully differentiate into mature dendritic cells, were associated with dexamethasone-mediated changes in CCL2 levels, and could be re-created in vitro using tumor supernatants. We provide evidence that tumors express high levels of CCL2, can contain high numbers of CD14(+) cells, that tumor supernatants can transform CD14(+)HLA-DR(+) cells into CD14(+)HLA-DR(lo/neg) immune suppressors, and that dexamethasone reduces CCL2 in vitro and is correlated with reduction of CCL2 in vivo. Consequently, we have developed a model for tumor mediated systemic immune suppression via recruitment and transformation of CD14(+) cells.

Immunosuppressive CD14+HLA-DRlow/- monocytes in prostate cancer.

OBJECTIVE: To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS: We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N = 18; mean age +/- SD: 72.1 +/- 6.9 years), tPCa (N = 22; 72.8 +/- 9.8 years) and age matched controls (AMC; N = 12; 68.8 +/- 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS: We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14(+)HLA-DR(low/-) monocytes, in tPCa (30.7 +/- 15.0% of CD14(+) cells) relative to AMC (4.1 +/- 6.5%, P < 0.0001) and uPCa (10.6 +/- 14.3%, P = 0.0001). The levels of CD14(+) HLA-DR(low/-) cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14(+)HLA-DR(low/-) cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14(+)HLA-DR(+) cells. CONCLUSIONS: This is the first report of CD14(+) cells exhibiting reduced expression of HLA-DR molecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

Demonstration of anti-tumor activity of oncolytic measles virus strains in a malignant pleural effusion breast cancer model.

Breast cancer is the second leading cause of malignant effusions in cancer patients. Pleural effusion indicates incurable disease with limited palliative treatment options and poor outcome. Here, we demonstrate the therapeutic efficacy of measles virus (MV) vaccine strain derivative against malignant pleural effusion in an MDA-MB-231 xenograft model of advanced breast cancer. Both systemic intravenous (i.v.) and intrapleural (t.t.) administered virus caused massive infection and syncytia formation in the pleural tumor deposits. Intrapleural administration of 1.5 x 10(6) plaque-forming units (PFU) total dose of MV significantly improved median survival by approximately 80% compared to the control animal group. Furthermore, we tested human dendritic cells as carriers for delivery of oncolytic MV infection to breast cancer pleural metastases. Carrier-delivered MV infection prevented accumulation of the pleural exudate and also significantly improved the survival of the treated mice. This is the first demonstration of the therapeutic potential of oncolytic virotherapy against malignant pleural effusions in a pre-clinical model of advanced breast cancer.

Dendritic cell microvilli: a novel membrane structure associated with the multifocal synapse and T-cell clustering.

Polarizing effects of productive dendritic cell (DC)-T-cell interactions on DC cytoskeleton have been known in some detail, but the effects on DC membrane have been studied to a lesser extent. We found that T-cell incubation led to DC elongation and segregation of characteristic DC veils to the broader pole of the cell. On the opposite DC pole, we observed a novel membrane feature in the form of bundled microvilli. Each villus was approximately 100 nm in diameter and 600 to 1200 nm long. Microvilli exhibited high density of antigen-presenting molecules and costimulatory molecules and provided the physical basis for the multifocal immune synapse we observed during human DC and T-cell interactions. T cells preferentially bound to this site in clusters often contained both CD4(+) and CD8(+) T cells.

A novel source of viable peripheral blood mononuclear cells from leukoreduction system chambers.

BACKGROUND: Buffy coats are becoming less available as a source of research-grade peripheral blood mononuclear cells (PBMNCs). Therefore, alternative sources of these cells were investigated. STUDY DESIGN AND METHODS: PBMNCs isolated from the cells retained in leukoreduction system chambers (LRSCs) and those eluted from white blood cell filters were compared. From LRSCs (1.88 +/- 0.40) x 10(9) PBMNCs (n = 13) versus (0.43 +/- 0.15) x 10(9) PBMNCs were isolated from leukofilter eluates (LFEs, n = 8; p < 0.0001). RESULTS: Cells from LRSCs and LFEs produced similar numbers of burst-forming unit-erythroid, colony-forming unit (CFU)-granulocyte-macrophage, and CFU-granulocyte-erythrocyte-monocyte-macrophage-megakaryocyte colonies. The percentages of cells positive for CD3, CD4, CD8, CD14, CD19, and CD56 in the PBMNCs isolated from LRSCs and LFEs were indistinguishable. Cells isolated from LRSCs expressed higher levels of CD69 and CD25 in reaction to staphylococcal enterotoxin B than the cells isolated from LFEs. The source of cells affected neither the yield and purity of immunomagnetically isolated CD3+ cells, CD14+ cells, and CD56+ cells nor the function of T cells, natural killer cells, and in vitro matured dendritic cells (DCs). DC yield from LRSC-derived CD14+ cells, however, was higher. CONCLUSION: LRSCs are a novel source of fully functional PBMNCs that can replace the more traditional sources of research-grade cellular products.

Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma.

BACKGROUND: Dendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines. METHODS: We prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma. RESULTS: Three preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 +/- 12 percent) in comparison to non-electroporated cells (82 +/- 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections. CONCLUSION: Standardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

Imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo.

Imatinib mesylate (STI571, imatinib) inhibited DNA synthesis in primary human T cells stimulated with allogeneic mature dendritic cells or phytohemagglutinin (PHA) but did not induce apoptosis. The values for the concentration that inhibits 50% (IC50) of T-cell proliferation stimulated by dendritic cells and PHA were 3.9 microM and 2.9 microM, respectively, that is, within the concentration range found in patients treated with imatinib mesylate. Interestingly, imatinib mesylate did not inhibit expression of T-cell activation markers CD25 and CD69, although it reduced the levels of activated nuclear factor-kappaB (NF-kappaB) and changed phosphorylation or protein levels of Lck, ERK1/2, retinoblastoma protein, and cyclin D3. When T cells were washed free of imatinib mesylate, they proliferated in response to PHA, demonstrating that inhibition is reversible. Treatment with imatinib mesylate led to accumulation of the cells in G0/G1 phase of the cell cycle. The in vitro observations were confirmed in vivo in a murine model of delayed-type hypersensitivity (DTH). In mice treated with imatinib mesylate, DTH was reduced in comparison to sham-injected controls. However, the number of splenic T cells was not reduced showing that, similarly to in vitro observations, imatinib mesylate inhibited T-cell response, but did not cause apoptosis. These findings indicate that long-term administration of high-dose imatinib mesylate might affect immunity.

Immunomagnetic separation reagents as markers in electron microscopy.

Antibodies coupled to magnetic particles have been employed for immunomagnetic cell isolation, but their consequent use for electron microscopy (EM) has not been evaluated. We used commercial antibodies coupled to iron-dextran to isolate T cells and monocytes/macrophages by immunomagnetic adsorption from normal human peripheral blood mononuclear cells. Subsequently, we studied the association of electron-dense immunomagnetic reagents with cell membranes. CD14-positive monocytes/macrophages isolated from fixed peripheral blood mononuclear cells retained electron-dense beads on the plasma membrane, while live cells internalized them. Flow cytometry and electron microscopy measurements of the percentage of cells that bound a CD4-specific immunomagnetic reagent in pan-T cell isolates (containing numerous T cell subtypes) were indistinguishable. The immunomagnetic reagent associated with cells could be secondarily labeled by secondary antibody coupled to colloidal gold. This study shows that these reagents used for cell isolation or just labeling, remain associated with their targets at the cell membrane. Immunomagnetic reagents allow "capturing" of rare cells from complex mixtures, purifying and concentrating them in a single step for subsequent electron microscopy. The large number of commercially available immunomagnetic reagents specific for different human, mouse and rat antigens provides additional resources for visualization of cellular ultrastructure.