Cells In Peripheral Blood Mononuclear Cells Research Articles

Magnetic Nanocarriers Enhance Drug Delivery Selectively to Human Leukemic Cells

Selective drug delivery to human leukemia cells using a nanoparticulate chemotherapeutic formulation is hugely needed. In this work, we report the development of a magnetic nanocarrier made of PVP-stabilized magnetic iron oxide nanoparticles (PMNP) loaded with the anticancer drug Doxorubicin (Dox) as a promising selective drug carrier to different types of human leukemia and normal cells. Our results revealed that while the unloaded MNPs were not potent to any of the cells, Dox@PMNPs showed significant toxicities, effectively killing the different leukemia cells, albeit at different inhibitory concentrations. Interestingly and superior to free Dox, Dox@PMNPs showed enhanced and significant inhibition towards the human monocytic THP-1 cells compared to human promyelocytic leukemia cells HL-60 (2-fold enhanced cytotoxicities), with the least potency towards the normal peripheral blood mononuclear cell (PBMC) cells (up to 6-fold). Nonetheless, free Dox was found to be concurrently less toxic to all the three cell lines tested. The cytotoxic effects obtained were further confirmed by live confocal imaging and electron microscopy. Both imaging techniques confirmed distinct morphological changes (membrane blebbing, shrinkage, and condensation) corresponding to typical apoptotic features in the treated leukemia cells compared to normal PBMC cells. The observed enhanced cytotoxic effects of Dox@PMNPs is mostly dependent upon the selective and differential endocytic uptake of Dox@PMNPs, with subsequent release of Dox intracellularly to the cytoplasm after 6 h, which then translocates to the nucleus after 24 h, causing apoptotic cell death. Importantly, magnetic Dox nanocarrier described here reduces the unwanted diffusive side effects of the free drug and allows selective drug delivery to leukemic cells, allowing its potential use for leukemic patients’ theranostics.

The Task of Desferrioxamine and Iron in the Immune Interplay between mononuclears and Colon Cancer Cells

Background: Macrophages and especially the tumor related ones play an essential role on malignant cell proliferation since they are tightly connected with iron absorption and release. The present study was designed to examine the role of iron and desferrioxamine on the immune interaction between human peripheral blood mononuclear cells (PBMC) and human colon carcinoma lines HT-29 and RKO.Materials and Methods: PBMC co-cultured with either HT-29 or RKO cells were depleted of iron by desferrioxamine (DFO). TNFα, IL-1β, IL-6, IFNγ, IL-2, IL-10 and IL-1ra concentration in the supernatants were examined by ELISA on various combinations of iron depleted and non-depleted PBMC and malignant cells.Results: DFO added to PBMC incubated with HT-29 cells, caused inhibited secretion of TNFα, IFNβ, IL-1ra and IL-10 whereas IL-1β, IL-6 and IL-2 production was not affected. Addition of DFO to PBMC cultured with RKO cells resulted in reduction of IL-1ra and IL-10 generation only. Depletion of iron from either PBMC or malignant cells and the addition of iron affected differently cytokine production.Conclusions: Both iron and DFO affect inflammatory cytokine production by human PBMC and intervene in the cross-talk between immune and colon cancer cells from two lines examined. The study enlightens the way iron and DFO may modulate tumorigenesis.

About the Role of Insulin in the Interaction Between Human Immune and Colon Cancer Cells

Background: Insulin has been one of the immense contributions to human health. Besides its role in treatment of diabetes, insulin affects inflammatory cytokine production, chronic inflammation and cancer development. We have examined the effect of insulin on cytokine production by human peripheral blood mononuclear cells (PBMC) and its role in the balance between immune and human colon cancer cells. Methods: Human LPS stimulated and non-stimulated PBMC, HT-29 and RKO cancer cell lines were separately incubated with insulin at various concentrations and the production of TNFα, IL-1β, IL-6, IFNγ, IL-1ra and IL-10 was examined. In addition, the effect of insulin on the secretion of these cytokines by PBMC co-incubated with carcinoma cells was evaluated. Results: Insulin added to non-stimulated PBMC caused a decreasaed secretion of the pro-inflammatory TNFα. Stimulation of PBMC with either LPS or PMA resulted in decreased production of IL-1β, TNFα and IFNγ and an increased generation of IL-6. The production of the anti-inflammatory cytokine IL-10 was elevated by both stimulated and unstimulated PBMC. Insulin added to PBMC inhibited the secretion of IFNγ stimulated with both HT-29 and RKO cells and reduced TNFα production induced by RKO cells. Conclusions: The results indicate that insulin interferes with inflammatory cytokine production by stimulated PBMC and it affects the cross talk between immune and colon cancer cells. It appears that the effect of insulin on the immune balance between mononuclear and malignant cells depends on its concentration and type of cancer cells. The findings provide and additional way for understanding the relation between insulin and cancerogenesis.

The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses.

Ebola virus (EBOV) infections are characterized by deficient T-lymphocyte responses, T-lymphocyte apoptosis and lymphopenia. We previously showed that disabling of interferon-inhibiting domains (IIDs) in the VP24 and VP35 proteins effectively unblocks maturation of dendritic cells (DCs) and increases the secretion of cytokines and chemokines. Here, we investigated the role of IIDs in adaptive and innate cell-mediated responses using recombinant viruses carrying point mutations, which disabled IIDs in VP24 (EBOV/VP24m), VP35 (EBOV/VP35m) or both (EBOV/VP35m/VP24m). Peripheral blood mononuclear cells (PBMCs) from cytomegalovirus (CMV)-seropositive donors were inoculated with the panel of viruses and stimulated with CMV pp65 peptides. Disabling of the VP35 IID resulted in increased proliferation and higher percentages of CD4+ T cells secreting IFNγ and/or TNFα. To address the role of aberrant DC maturation in the IID-mediated suppression of T cell responses, CMV-stimulated DCs were infected with the panel of viruses and co-cultured with autologous T-lymphocytes. Infection with EBOV/VP35m infection resulted in a significant increase, as compared to wt EBOV, in proliferating CD4+ cells secreting IFNγ, TNFα and IL-2. Experiments with expanded CMV-specific T cells demonstrated their increased activation following co-cultivation with CMV-pulsed DCs pre-infected with EBOV/VP24m, EBOV/VP35m and EBOV/VP35m/VP24m, as compared to wt EBOV. Both IIDs were found to block phosphorylation of TCR complex-associated adaptors and downstream signaling molecules. Next, we examined the effects of IIDs on the function of B cells in infected PBMC. Infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in significant increases in the percentages of phenotypically distinct B-cell subsets and plasma cells, as compared to wt EBOV, suggesting inhibition of B cell function and differentiation by VP35 IID. Finally, infection with EBOV/VP35m increased activation of NK cells, as compared to wt EBOV. These results demonstrate a global suppression of cell-mediated responses by EBOV IIDs and identify the role of DCs in suppression of T-cell responses.

A Novel Dendritic Cell-Derived Vaccine in Multiple Myeloma

Introduction: Despite recent advances in the treatment of multiple myeloma (MM), curative outcomes remain elusive with standard therapies. MM is characterized by immune dysregulation and a loss of tumor-associated T cell surveillance contributing to disease evolution. Cancer vaccines have emerged as a promising immunotherapeutic strategy that seeks to reestablish anti-tumor immunity through the effective presentation of tumor associated antigens in the context of positive costimulation and activation signals. DCOne is a cell based tumor vaccine derived from an acute myelogenous leukemia cell line, differentiated toward a dendritic cell (DC) phenotype (DCPrime, Netherlands). DCOne strongly expresses WT-1, PRAME, RHAMM, survivin, and MUC-1. Since MM cells commonly express these tumor-associated antigens as well, we explored the efficacy of DCOne in inducing myeloma-specific immunity.Methods/Results: We first investigated the capacity of DCOne to polarize T cells toward an activated phenotype. Peripheral blood mononuclear cells (PBMCs) derived from MM patients were cultured in the presence or absence of DCOne and pulsed with whole cell DCOne lysate 24 hours prior to analysis. Intracellular levels of IFN-γ and perforin in CD8+ T cells and intracellular levels of IL-10 in CD4+ T cells were assessed via FACS analysis. Following 10 days of co-culture, we observed an increased percentage of CD8+ IFN-γ+ T cells and an increased percentage of CD8+ perforin+ T cells in PBMCs co-cultured with DCOne versus those not co-cultured with DCOne (10.8% versus 3.3%, p=0.02, n=11 and 6.6% versus 1.7%, p=0.045, n=10, respectively). In contrast, exposure of PBMCs to DCOne did not alter the percentage of CD4+ IL-10+ T cells (p=0.53, n=8). Given the observed increase in IFN-γ- and perforin-positive CD8+ T cells after co-culture of MM PBMCs with DCOne, we investigated whether CD8+ T cells co-cultured with DCOne exhibited enhanced killing of autologous tumor cells in a standard cytotoxic T lymphocyte (CTL) assay measuring Granzyme B activity by FACS analysis. DCOne potently induced CTL-mediated killing of autologous MM cells as determined by an increased percentage of CD8+ Granzyme B+ cells in stimulated versus control cells (27.4% versus 12.5%, p=0.05, n=3).We next investigated the mechanism of action by which DCOne elicited a MM-specific response. We postulated that the allogeneic cell line would induce immune activation in part through the transfer of antigen to PBMC-derived antigen presenting cells; these antigen presenting cells would in turn be capable of eliciting response via MHC restricted presentation of antigen to autologous T cell populations. Consistent with this hypothesis, we demonstrated that 32% of patient-derived PBMCs exhibited uptake of DCOne RNA following co-culture (n=3). Uptake of DCOne RNA by patient-derived PBMCs was completely abrogated with Transwell separation. Next, we examined how patient-derived PBMCs take up DCOne antigens. Using Western Blot analysis, we found that MUC-1 and survivin are expressed in extracellular vesicles (EVs) derived from DCOne lysate. Thus, we conclude that DCOne antigens are trafficked via EVs secreted by the DCOne parent cell, whereupon they are taken up by MM patient-derived PBMCs.Conclusion: In conclusion, when cultured in vitro with MM patient-derived PBMCs, DCOne results in increased IFN-γ- and perforin-positive CD8+ T cells, as well as induction of autologous tumor killing by these cells. DCOne RNA is taken up by MM patient-derived PBMCs and trafficked to the extracellular environment via EVs. DCOne demonstrates in vitro efficacy as an "off-the-shelf" strategy for stimulating MM-specific immunity. A clinical trial is being planned. DisclosuresKruisbeek:DCPrime: Employment, Other: Founder, CSO, and CEO of DCPrime. Van Wetering:DCPrime: Employment. Arnason:Gilead: Consultancy. Rosenblatt:DCPrime: Research Funding; BMS: Research Funding; Astex: Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding.

Capsaicin Modulates the Immune Cross Talk Between Human Mononuclears and Cells from Two Colon Carcinoma Lines

ABSTRACTBackground: Capsaicin, the pungent alkaloid of the chili peppers, has gained a worldwide reputation. In addition to its culinary assets, capsaicin possesses analgesic, anti-inflammatory, antimicrobial, and even carcinopreventive properties. Considering the linkage between chronic inflammation and tumorigenesis, the aim of the study was to evaluate the role of capsaicin in the immune interplay between human peripheral blood mononuclear cells (PBMCs) and HT-29 or RKO cells from human colon carcinoma lines. Methods: PBMCs were incubated for 24 hours with either HT-29 or RKO cells and concentrations of capsaicin ranging between 10 and 200 µM. Subsequently, the generation of the following cytokines was examined: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10. Results: Capsaicin caused a concentration-dependent inhibition of colon cancer cells proliferation but had no effect on PBMC viability. 200 µM of capsaicin suppressed the production of all cytokines tested. At lower concentrations, the secretion of TNF-α, IL-1β, IFN-γ, IL-10, and IL-1ra was inhibited concentration-dependently, whereas that of IL-6 was stimulated. Conclusions: Capsaicin causes a concentration-dependent alteration of the immune balance between PBMC and colon carcinoma cells expressed as an inhibited generation of inflammatory cytokines. These findings indicate the existence of an additional immunomodulatory mechanism by which this alkaloid may prevent tumor development.

Upregulated Expression of microRNA-16 Correlates with Th17/Treg Cell Imbalance in Patients with Rheumatoid Arthritis

To explore the correlation between miR-16 expression in T cells of peripheral blood mononuclear cells (PBMCs) and Th17/Treg imbalance in rheumatoid arthritis (RA) patients. Forty RA patients were recruited as the case group and further grouped as active RA and inactive RA groups; 21 healthy individuals were selected as the control group. Th17 and Treg were measured by flow cytometry, and their related cytokines were measured by FlowCytomix. RORγt, FoxP3 mRNA, and miR-16 expression in T cells was determined by real-time quantitative polymerase chain reaction. Western blotting was performed to measure RORγt and FoxP3 protein expression. RA patients showed upregulated Th17 and RORγt mRNA and protein expression compared with the controls (all p < 0.05); active RA patients showed lower Treg and FoxP3 mRNA and protein expression compared with inactive RA patients and controls (all p < 0.05). Secretion levels of Th17-related cytokines were higher in active RA patients than in inactive RA patients and controls (all p < 0.05); whereas those of Treg-related cytokines were lower in active RA patients than in controls (all p < 0.05). Active RA patients showed increased miR-16 expression in Th17 cells and decreased miR-16 expression in Treg cells of PBMCs (both p < 0.05). Pearson's test showed that in the PBMCs of the RA patients, miR-16 expression in the Th17 cells was positively related with RORγt mRNA expression, and miR-16 expression in the Treg cells was positively related with FoxP3 mRNA expression (both p < 0.05). The expression of miR-16 in Th17 and Treg cells of PBMCs in RA patients was closely associated with the expression of RORγt and FoxP3. MiR-16 may be involved in Th17/Treg imbalance of RA patients by affecting the expression of RORγt and FoxP3.

Heterogeneity of Specific CD4+ and CD8+ T Cells Stimulated by CMV pp65 and IE1 Antigens.

Characterization of human cytomegalovirus-specific T cells (CMV-T) is of critical importance for their potential use in adoptive immunotherapy after allogeneic hematopoietic stem cell transplantation. Background frequencies of CMV-T in peripheral blood mononuclear cells (PBMCs) of CMV-seropositive healthy subjects are usually very low, hence the requirement for prolonged culture time and multiple stimulations to expand them. The evaluation of the end-culture specificity and composition has sometimes been neglected or difficult to assess in these settings. We explored the identity and the functionality of pp65-specific and IE1-specific T cells, enriched in short-term cultures from PBMCs. Antigen-specific T cells were further isolated by IFN-γ capture system and/or CD154 microbeads. Frequency of IE1-specific cytotoxic T cells in PBMCs secreting IFN-γ was higher compared with the pp65-specific one, whereas the latter cell types showed a higher median CD107a degranulation. Cell viability, rate of CMV-T increase, and multicytokine secretion profile after epitope-specific short-term cultures were heterogenous. T cells were mainly of late effector stages but they significantly dropped off upon CMV rechallenge with peptide pools. In parallel, CMV-T expansion was accompanied by a significant increase of cytotoxic naive/memory stem cells (CTLs), whereas the CD4 counterpart significantly increased only upon stimulation with IE1. Outcome was variable and showed donor and epitope dependency. Differences in human leukocyte antigen and epitope dominance and variability in the relative number of CD3 effector cells and IFN-γ/CD154 expression among healthy donors could reflect the observed individual CMV-specific cellular immunity. This heterogeneity raises points to be considered when approaching adoptive immunotherapy.

Rituximab induces phenotypical and functional changes of NK cells in a non-malignant experimental setting.

BackgroundRituximab has broad and increasing application in rheumatic diseases. It is known from lymphoma studies that natural killer (NK) cells can lyse rituximab-coated transformed B cells. However, the role of NK cells in mediating rituximab-induced depletion of non-malignant B cells is unknown. The purpose of this study was to provide fundamental data on rituximab-mediated effects on NK cells in PBMCs without tumor cells, in order to simulate effects that could be relevant in patients with rheumatic disease.MethodsFreshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured overnight with therapeutic antibodies. NK cells were isolated using a commercial kit or depleted from PBMCs using anti-CD56 and anti-CD16 monoclonal antibodies and magnetic beads. Cells were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using 51Cr-labeled K562 target cells.ResultsAddition of rituximab to PBMCs resulted in depletion of B cells, which was dependent on NK cells and serum factors. The extent of B cell depletion correlated with the percentage of NK cells. Following incubation with rituximab, NK cells within PBMCs were activated, degranulated and downregulated the low affinitiy Fc-γ-receptor CD16 (FcγRIIIA). The co-activating receptor CD137 (41BB) was upregulated on a fraction of NK cells. NK cell function was altered in some donors in whom we observed rituximab-dependent reduction in NK cell cytotoxicity towards K562 tumor cells.ConclusionsNK cells mediate rituximab-induced B cell depletion. Rituximab induces altered NK cell phenotype and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1101-3) contains supplementary material, which is available to authorized users.

IL-9-producing Th9 cells may participate in pathogenesis of Takayasu’s arteritis

Takayasu's arteritis (TAK) is a type of large vessel vasculitis which involves the aorta and its major branches. Interleukin (IL)-9 or IL-9-producing Th9 cells were found to be involved in pathogenesis of autoimmune arteritis such as giant cell arteritis, but IL-9 or Th9 cells in TAK were not well known. Here, this study aims to analyze the levels of serum IL-9 and their major source Th9 cells in TAK. With the help of cytometric bead array (CBA), a total of 21 patients with TAK were examined for serum levels of cytokines IL-4, IL-6, IL-8, IL-17, IL-10, TNF-α, IFN-γ, and IL-9. Flow cytometry techniques were used to examine the frequencies of Th9 cells from peripheral blood mononuclear cells (PBMCs) for 11 patients with active TAK and 10 healthy controls. Higher serum levels of serum IL-6 (P<0.05), TNF-α (P<0.05), and IL-9 level (P<0.05) were observed in TAK patients compared to those of healthy controls. Higher frequencies of CD4+ IL-9+ T cells and CD4+ PU.1+ T cells in PBMCs and IL-9+ PU.1+ T cells in CD4+ T cells were observed in active TAK patients than those in healthy controls (all P<0.01). The levels of IL-9 had a positive correlation with ESR (r=0.975, P=0.015) in these cases. Our data suggested that Th9 cells and IL-9 could possibly be involved in the pathogenesis of TAK.

Assay of Peripheral Regulatory Vδ1 T Cells in Ankylosing Spondylitis and its Significance.

BackgroundAnkylosing spondylitis (AS) involves inflammation at the sacroiliac joint and spine attachment site. This study aimed to observe the ratio and function of peripheral regulatory Vδ1 T cells in AS patients to investigate their roles in AS pathogenesis.Material/MethodsPeripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation from AS patients and healthy controls. Flow cytometry was used to determine the ratio between Vδ1 and CD4 T cells of PBMC in AS patients and controls. Flow cytometry sorting (FCS) was used to obtain Vδ1 and naïve CD4 T cells with purity higher than 90%. CFSE staining method was used to detect the effect of Vδ1 T cells on proliferation of naïve CD4 T cells. The effect of Vδ1 T cells on secretion of IFN-γ from naïve CD4 T cells and the ability to secrete IL-10 from Vδ1 T cells were determined by flow cytometry.ResultsAS patients had significantly lower Vδ1 T cell ratio in PBMC compared to controls (p<0.05), but their CD4 T cell ratio was significantly elevated (p<0.05). Functional assay showed suppression of naïve CD4 T cell proliferation and IFN-γ secretion by peripheral Vδ1 T cells in AS patients (p<0.01). AS patients also had lower IL-10 secreting level from peripheral derived Vδ1 T cells (p<0.01).ConclusionsThe immune suppression of peripheral Vδ1 T cell in AS patient increases the ratio of peripheral CD4 T cells and IFN-γ level, leading to AS pathogenesis. This immune suppression is mainly due to suppressed IL-10 secretion.

Macrophage interactions with polylactic acid and chitosan scaffolds lead to improved recruitment of human mesenchymal stem/stromal cells: a comprehensive study with different immune cells.

Despite the importance of immune cell-biomaterial interactions for the regenerative outcome, few studies have investigated how distinct three-dimensional biomaterials modulate the immune cell-mediated mesenchymal stem/stromal cells (MSC) recruitment and function. Thus, this work compares the response of varied primary human immune cell populations triggered by different model scaffolds and describes its functional consequence on recruitment and motility of bone marrow MSC. It was found that polylactic acid (PLA) and chitosan scaffolds lead to an increase in the metabolic activity of macrophages but not of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells or monocytes. PBMC and NK cells increase their cell number in PLA scaffolds and express a secretion profile that does not promote MSC recruitment. Importantly, chitosan increases IL-8, MIP-1, MCP-1 and RANTES secretion by macrophages while PLA stimulates IL-6, IL-8 and MCP-1 production, all chemokines that can lead to MSC recruitment. This secretion profile of macrophages in contact with biomaterials correlates with the highest MSC invasion. Furthermore, macrophages enhance stem cell motility within chitosan scaffolds by 44% but not in PLA scaffolds. Thus, macrophages are the cells that in contact with engineered biomaterials become activated to secrete bioactive molecules that stimulate MSC recruitment.

Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis

The present study evaluated associations between the percentages of T cell immunoglobulin and mucin domain 3 (Tim3)-positive T cells and related cytokines and multiple sclerosis (MS). We collected peripheral blood samples from 30 MS patients and 30 healthy controls. Flow cytometry was used to determine the proportions of CD3+Tim3+, CD4+Tim3+, and CD4+CD25+Tim3+ in peripheral blood mononuclear cells (PBMCs) and related cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ also were determined using enzyme-linked immunosorbent assays (ELISA). The percentages of Tim3-positive T cells in CD4+ and CD4+CD25+ T cell subsets were significantly lower among MS patients than among controls. This difference was particularly evident in the CD4+CD25(high) T cell subset. The proportions of CD4+Tim3+ and CD4+CD25+Tim3+ cells in PBMCs were significantly lower in the MS group than in the control group, whereas no significant differences were detected regarding the percentages of CD3+Tim3+ in PBMCs and T cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ all were increased in MS patients compared with healthy controls. Our results support that Tim3 and related cytokines may be involved in the onset of MS.

Role of podoplanin in the high interleukin-17A secretion resulting from interactions between activated lymphocytes and psoriatic skin-derived mesenchymal cells.

In the context of psoriasis, T helper type 17 (Th17) cells infiltrate the inflammatory site and interact with local mesenchymal cells, including skin fibroblasts. The aim of this work was to study the interactions of skin-derived fibroblasts with peripheral blood mononuclear cells (PBMC) with a focus on the Th17 pathway and to identify a mechanism which leads to a high interleukin (IL)-17 secretion. A co-culture system between PBMC and skin fibroblasts was developed. Healthy and patient PBMC were added to non-lesional or lesional skin fibroblasts at a 5:1 ratio for 48 h in the presence or not of activation with phytohaemagglutinin (PHA). Monocytes were removed or not by adherence before the co-culture. An anti-podoplanin antibody was also used during the co-culture. Cytokine production (IL-8, IL-6, IL-1β and IL-17) was measured by enzyme-linked immunosorbent assay (ELISA) and cell staining (CD3, CD4, IL-17 and podoplanin) by flow cytometry. Without T cell receptor (TCR) activation, IL-8, IL-6 and IL-1β production increased in PBMC-fibroblast co-culture compared to PBMC alone. No additional effect was observed with TCR activation, with no difference in the Th17 cell percentage in activated-PBMC alone or co-cultured. Conversely, IL-17 production was increased highly only in co-cultures between control and patient activated-PBMC and skin fibroblasts. Removal of monocytes decreased cytokine production, notably that of IL-17. Addition of an anti-podoplanin antibody decreased IL-17 secretion by 60%. Interactions between resting PBMC and fibroblasts induce the IL-8, IL-6 and IL-1β production. PBMC activation and cell interactions are critical for a high IL-17 secretion. Podoplanin contributes largely to this massive IL-17 secretion.

The impact of intestinal resection on the immune function of short bowel syndrome patients

Short bowel syndrome (SBS) is characterized by a massive intestinal loss after surgery resection. Likewise, disturbances involving the intestine, which represents a complex immune environment, may result in breakdown of homeostasis and altered responses, thus leading to unpredictable clinical outcomes. However, the consequences of bowel resection were poorly investigated until now. Therefore, this study aimed to evaluate the immunological status of SBS-patients. For this purpose, ten subjects and nine healthy controls were evaluated. Along with some metabolic disturbances, the main results showed higher levels of the inflammatory cytokine IL-6 in plasma among SBS-patients. However, there were no differences in the frequency of CD3+, CD3+CD4+ or CD3+CD8+ T lymphocytes. An augmented frequency in CD4+ and CD8+ cells producing IFN-γ was also observed in peripheral blood mononuclear cells (PBMC), together with elevated percentage of CD4+ cells producing IL-10. No differences were observed in the frequency of total CD4+CD25−, CD4+CD25+ lymphocytes nor in the expression of FoxP3 or GITR. Nevertheless, SBS-patients showed higher frequency of the regulatory T cell population CD4+CD25+CD39+ cells in PBMC. In conclusion, these data pointed to SBS as an important disturbance that compromises not only the intestinal environment but also negatively influences systemic immune components.

Effects of active bufadienolide compounds on human cancer cells and CD4+CD25+Foxp3+ regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

The growth inhibitory effects of bufadienolide compounds were investigated in two intractable cancer cells, a human glioblastoma cell line U-87 and a pancreatic cancer cell line SW1990. Among four bufadienolide compounds, a dose-dependent cytotoxicity was observed in these cancer cells after treatment with gamabufotalin and arenobufagin. The IC50 values of the two compounds were 3-5 times higher in normal peripheral blood mononuclear cells (PBMCs) than these values for both cancer cell lines. However, similar phenomena were not observed for two other bufadienolide compounds, telocinobufagin and bufalin. These results thus suggest that gamabufotalin and arenobufagin possess selective cytotoxic activity against tumor cells rather than normal cells. Moreover, a clear dose-dependent lactate dehydrogenase (LDH) release, a well-known hallmark of necrosis, was observed in both cancer cells treated with gamabufotalin, suggesting that gamabufotalin-mediated cell death is predominantly associated with a necrosis-like phenotype. Of most importance, treatment with as little as 8ng/ml of gamabufotalin, even an almost non-toxic concentration to PBMCs, efficiently downregulated the percentages of CD4+CD25+Foxp3+ regulator T (Treg) cells in mitogen-activated PBMCs. Given that Treg cells play a critical role in tumor immunotolerance by suppressing antitumor immunity, these results suggest that gamabufotalin may serve as a promising candidate, as an adjuvant therapeutic agent by manipulating Treg cells to enhance the efficacy of conventional anticancer drugs and lessen their side-effects. These findings provide insights into the clinical application of gamabufotalin for cancer patients with glioblastoma/pancreatic cancer based on its cytocidal effect against tumor cells as well as its depletion of Treg cells.

Abstract 4138: Immunological profile of metastatic or recurrent breast cancer patients

Abstract Background: We previously reported about immune suppression in breast cancer patients at this meeting. In brief, tumor tissue specimens and peripheral blood mononuclear cells (PBMC) were analyzed in 50 early or advanced breast cancer (BC) patients. To compare between 38 early and 12 advanced BC cases, CD163-positive tumor cells and CCR4-positive tumor cells were detected more frequently in advanced cases than in early cases. Regulatory T (Treg) cells in PBMC significantly increased in percentage of the population in 37 BC patients than in 21 healthy volunteers (AACR 2011). In addition, several cytokines were examined in that cohort and plasma IL-17A had significantly higher levels in early BC than in advanced BC (AACR 2013). Then, we examined their prognosis and immunological profile. Patients and methods: Treg cells were examined by counting CD4+CD25highCD127low/-cells in PBMC with flow cytometry analysis. Immunohistochemical evaluation of tumor specimens was performed with monoclonal antibodies of HLA-ABC and DR, CD56, CD68, CD83, CD163 and CCR4. The number of stained cells was analyzed using a semiquantitative ordinal scale ranging from 0 to 3 (0, +/-, ++, +++). Human IL-2, IL-4, IL-6, IL-10, TNF, INFγ and IL-17A were measured using cytometric beads array system. Most patients received chemotherapy, hormonal therapy, and/or anti-HER2 therapy on the basis of intrinsic subtype and breast irradiation after breast-conserving surgery. Results and discussions: At the median follow-up of 7 years after blood sample collection, only 2 operable patients relapsed and 3 patients including 2 cases of stage IV died of disease. Of 5 cases of stage IV or recurrent BC, CD163 and/or CCR4 were strongly stained positive in tumor cells. There were significant differences of staining intensity in CD163 and CCR4-positive tumor cells between those cases and the rest 45 cases (p&amp;lt;0.01 at Chi-square test). However, other immune cell profiles, Treg cells in PBMC and cytokines in plasma had no trend between them. Several reports demonstrated that cancer patients with CD163-positive tumor-infiltrating macrophages and CD163-positive tumor cells had poor prognosis due to tumor-associated macrophage. Phenotypic macrophage traits in cancer cells, like CD163 expression, may be explained by heterotypic cell fusion between monocytes/macrophages and cancer cells. Conclusion: Targeted therapy against M2 macrophage or CCR4 is considered as a promising strategy of advanced breast cancer. Citation Format: Shigeru Imoto, Takayuki Ueno, Hirotsugu Isaka, Hiroki Ito, Kaisuke Miyamoto, Tomohiro Chiba, Hiroshi Kamma. Immunological profile of metastatic or recurrent breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4138.

Abstract 4154: Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer

Abstract Tumor-infiltrating immune cells (TIIs) in HER2+ breast cancer (BC) play an important role in treatment with anti-HER2 therapy. However, the precise mechanisms of how TIIs exert anti-tumor activity remain unclear. Neuropilin-1 (NRP-1) on macrophages regulates immune functions in various cancers and thus we explored the role of NRP-1 expressing macrophages in anti-tumor activity in HER2+ BC. We show that NRP-1 on macrophages regulated the migration of and chemokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model showed that NRP-1 knockdown of macrophages in adoptively transferring peripheral blood mononuclear cells (PBMCs) suppressed anti-tumor activity and infiltration of CD45+ immune cells into tumors. Interestingly, NRP-1 expressing TIIs were mainly CD4+ T cells, despite little expression of NRP-1 on CD4+ T cells in PBMCs. We found that NRP-1 expression on CD4+ T cells was induced by NRP-1 transfer from macrophages to T cells. In HER2+ BC patients, NRP-1 expressing TIIs correlated with better clinical outcomes. These results demonstrate that NRP-1 expressing macrophages are key subsets of immune cells in trastuzumab-mediated anti-tumor activity and may predict better outcomes for HER2+ BC patients. In conclusion, NRP-1 expression is required for differentiation and activation of macrophages. NRP-1 expressing macrophages promote NRP-1 expression on CD4+ T cells by direct interaction and contribute to anti-tumor immune responses in HER2+ BC. Citation Format: Kosuke Kawaguchi, Eiji SUzuki, Masashi Inoue, Isao Kii, Tatsuki R. Karaoke, Masahiro Hiram, Keiko Iwaisako, Pu Fengling, Mariko Niche, Ayane Yamaguchi, Hironori Haga, Masatoshi Hagiwara, Masakazu Toi. Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4154.

Interaction among activated lymphocytes and mesenchymal cells through podoplanin is critical for a high IL-17 secretion

BackgroundDuring chronic inflammation, immune cells, notably Th17 cells, infiltrate the inflammatory site and interact with local mesenchymal cells. Applied to rheumatoid arthritis (RA), the aim is to study the interactions between synoviocytes and peripheral blood mononuclear cells (PBMC) with a focus on the Th17 pathway and to identify a mechanism which leads to high IL-17 secretion with an interest on podoplanin.MethodsPBMC from healthy donors and RA patients were co-cultured with RA synoviocytes during 48 h, in the presence or not of phytohemagglutinin. An antibody against podoplanin was used in co-culture. Cytokine production (IL-6, IL-1β and IL-17) was measured by ELISA and cell staining (CD3, CD4, IL-17 and podoplanin) by flow cytometry.ResultsIn control conditions, IL-6 and IL-1β production was increased in PBMC-synoviocyte co-culture compared to PBMC alone (p = 0.02). No additional effect was observed with PBMC activation. Flow cytometry analysis showed no difference in the percentage of Th17 cells in activated PBMC alone or with synoviocytes (p = 0.4), indicating that Th17 differentiation requires only T cell activation. Conversely, IL-17 production was highly increased in co-cultures with activated PBMC vs. activated PBMC alone (p = 0.002). Transwell experiments confirm that cell-cell contact was critical for IL-17 secretion. The incubation of either PBMC or synoviocytes with an anti-podoplanin antibody decreased IL-17 secretion by 60 % (p = 0.008).ConclusionsInteractions between resting PBMC and synoviocytes are sufficient to induce IL-6 and IL-1β production. Both PBMC activation and cell interactions are needed to induce a high IL-17 secretion. Podoplanin contributes at the level of both lymphocytes and synoviocytes.

Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo.