Cytokine Detection Assay Research Articles

Plasma-derived exosomal protein SHP2 deficiency induces neutrophil hyperactivation in Behcet's uveitis

To investigate the effect of plasma-derived exosomal proteins on neutrophil hyperactivation in Behcet's uveitis (BU), we treated neutrophils from healthy controls with plasma-derived exosomes from active BU patients, and determined the level of neutrophil activation by real-time quantitative PCR (RT-qPCR) and cytokine detection assay. The results revealed that exosomes from active BU patients could activate neutrophils as shown by increasing the expression levels of pro-inflammatory cytokines (IL-17 and IL-6), chemokines (IL-8 and MCP-1), and NETs (MPO and ELANE). Label-free quantitative proteomic analysis of plasma-derived exosomes from patients and healthy controls found a remarkably distinct protein profile and identified differentially expressed proteins (DEPs) between the two groups. The results of GO, KEGG, and GSEA enrichment analysis showed that DEPs were enriched in innate immune-mediated and neutrophil hyperactivation-related signaling pathways. The protein-protein interaction (PPI) analysis determined that SHP2 was a downregulated key hub protein in the exosomes of active BU patients. Knockdown of SHP2 in human neutrophil cell lines (NB4 cells) was shown to promote the secretion of pro-inflammatory cytokines, chemokines, and NETs. The converse effects were observed following SHP2 overexpression. In conclusion, we highlighted a pathogenic role of plasma-derived exosomal SHP2 deficiency in facilitating neutrophil activation and suggested that SHP2 might be an immunoprotective factor in BU pathologic process.

Secretory IgA-ETEC F5 Immune Complexes Promote Dendritic Cell Differentiation and Prime T Cell Proliferation in the Mouse Intestine.

Although secretory IgA (SIgA) is the dominant antibody in mucosal secretions, the capacity of the SIgA-antigen complex to prime the activation of dendritic cells (DCs) and T cells in the intestinal epithelium is not well understood. To this end, the SIgA-ETEC F5 immune complexes (ICs) were prepared via Ni-NTA pull-down. After injecting the ICs into the intestines of SPF BALB/c mice, most ICs were observed in the Peyer's patch (PP). We established a microfold (M) cell culture model in vitro for transport experiments and the inhibition test. To evaluate the priming effect of mucosal immunity, we employed the DC2.4 stimulation test, T lymphocyte proliferation assays, and cytokine detection assays. We found that the ICs were taken up via clathrin-dependent endocytosis through M cells. The high expression of costimulatory molecules CD86, CD80, and CD40 indicated that the ICs promoted the differentiation and maturation of DC2.4 cells. The stimulation index (SI) in the complex group was significantly higher than in the control group, suggesting that the ICs stimulated the proliferation of primed T cells. The secretion of some cytokines, namely TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6, in spleen cells from the immunized mice was upregulated. These results indicate that ETEC F5 delivery mediated by SIgA in PPs initiates mucosal immune responses.

The meta-analysis for ideal cytokines to distinguish the latent and active TB infection

BackgroundOne forth whole-world population is infected with Mycobacterium tuberculosis (Mtb), but 90% of them are asymptotic latent infection without any symptoms but positive result in IFN-γ release assay. There is lack of ideal strategy to distinguish active tuberculosis (TB) and latent tuberculosis infection (LTBI). Some scientist had focused on a set of cytokines as biomarkers besides interferon- gamma (IFN-γ) to distinguish active TB and LTBI, but with considerable variance of results. This meta-analysis aimed to evaluate the overall discriminative ability of potential immune molecules to distinguish active TB and LTBI.MethodsPubMed, the Cochrane Library, and Web of Science databases were searched to identify studies assessing diagnostic roles of cytokines for distinguishing active TB and LTBI published up to August 2018. The quality of enrolled studies was assessed using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2). The pooled diagnostic sensitivity and specificity of each cytokine was calculated by using Meta-DiSc software. Area under the summary receiver operating characteristic curve (AUC) was used to summarize the overall diagnostic performance of each biomarker.ResultsFourteen studies with 982 subjects met the inclusion criteria, including 526 active TB and 456 LTBI patients. Pooled sensitivity, specificity and AUC for discriminating between active TB and LTBI were analyzed for IL-2 (0.87, 0.61 and 0.9093), IP-10 (0.77, 0.73 and 0.8609), IL-5 (0.64, 0.75 and 0.8533), IL-13 (0.75, 0.71 and 0.8491), IFN-γ (0.67, 0.75 and 0.8031), IL-10 (0.68, 0.74 and 0.7957) and TNF-α (0.67, 0.64 and 0.7783). The heterogeneous subgroup analysis showed that cytokine detection assays, TB incidence, and stimulator with Mtb antigens are main influence factors for their diagnostic performance.ConclusionsThe meta-analysis showed cytokine production could assist the distinction between active TB and LTBI, IL-2 with the highest overall accuracy. No single biomarker is likely to show sufficiently diagnostic performance due to limited sensitivity and specificity. Further prospective studies are needed to identify the optimal combination of biomarkers to enhanced diagnostic capacity in clinical practice.

Abstract 187: Trastuzumab induced myeloid cell alterations identifies mechanistic properties of vascular normalization

Abstract Introduction: The purpose of this study is to quantify longitudinal changes in myeloid cell infiltration after trastuzumab treatment to reveal mechanistic properties of improved tumor vascularization in human epidermal growth factor receptor type 2 positive (HER2+) breast cancer. We have previously presented in vivo data showing that trastuzumab increases vascular maturation leading to better delivery of therapeutics. The balance of myeloid cells in the tumor microenvironment (TME) can impact neovascularization. Specifically, M2 macrophages (MACs) promote angiogenesis causing irregular vascularization and M1 MACs exhibit anti-angiogenic properties counteracting the M2 cells and improving tumor perfusion. We present results revealing macrophage polarization towards an M1 phenotype after trastuzumab therapy in HER2+ tumors. Experimental Design: 107 BT474 breast cancer cells were implanted subcutaneously into athymic nude mice. After tumors reached 250 mm3, mice were treated with trastuzumab (10 mg/kg) or saline over one week and were excised for analysis on days 0, 4 and 7. Half of each tumor was fixed in 10% neutral buffered formalin for immunohistochemical staining (IHC) of the TME and half was digested for analysis of myeloid cell infiltration with flow cytometry. The flow cytometry panel used consists of ten markers, two specifically for the identification of M1 and M2 MACs; CD38, and CD206, respectively. A non-parametric Wilcoxon rank sum test was used to determine statistical differences between control and treated groups. Results and Discussion: Initial studies revealed an increase (P = 0.03) of MACs in day 4 and day 7 treated tumors. Of these MACs, there was an increase (P = 0.03) in co-expression of M2 and M1 phenotype markers CD206 and CD38 in day 4 treated tumors indicating a phenotypic switch occurring. After a third dose of trastuzumab, there was an increase (P = 0.03) in M1 MACs (CD38+/CD206-) in day 7 treated tumors. In day 4 and 7 treated tumors, there was a decrease (P = 0.02) in non-differentiated MACs (CD38-/CD206-) supporting evidence that a phenotypic switch is occurring. Ongoing studies are evaluating spatial variations in MAC infiltration (CD206, F4/80), hypoxia (pimonidazole), and vascular maturation index (ratio of alpha-smooth muscle actin to total vessel counts as stained with CD31) through IHC. Cytokine detection assays are being done to evaluate changes in cytokines contributing to macrophage polarization and angiogenesis. Conclusion: These results offer strong evidence supporting the hypothesis that MACs in the TME are switching to an M1 phenotype after trastuzumab treatment and contributing to trastuzumab induced vascular alterations. Analyses of IHC will validate vascular changes occurring simultaneously with myeloid infiltratration. Citation Format: Meghan J. Bloom, Angela M. Jarrett, Todd A. Triplett, Anum K. Syed, Thomas E. Yankeelov, Anna G. Sorace. Trastuzumab induced myeloid cell alterations identifies mechanistic properties of vascular normalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 187.

The role of stem cell metabolites derived from placenta for skin regeneration: An in vitro study

Background: The role of stem cells in skin aging is to repair injured tissue or replace other cells in programmed cell death. Stem cell metabolites are rich in growth factors including IL-10, IL-4, EGF, GM-CSF, and TGF-β that can induce the skin production of protein and elastic fbers, leading to the improvement of skin appearance. Aim: This study aimed to assess the characteristics of stem cell metabolites in vitro. Methods: Cytotoxicity assay was performed using MTT reagents and optical cell densities were determined using ELISA reader to fnd the percentage of living cells. Cytokine detection assay was performed by analyzing the cytokine levels in the peripheral blood mononuclear cell (PBMC) and mesenchymal stem cell (MSC) using ELISA. Apoptosis assay was performed using the double staining method with the markers identifed were Hsp70, p53, and caspase-3. Results: All samples showed the percentage of living cells that exceed 70%. Cytokine detection assay showed a decrease of IL- 12 and IFN-γ in both PBMC and MSC groups. The apoptosis assay of human adipose mesenchymal stem cells using a fluorescence microscope showed most of the green light was lost in control cells without metabolites. We found that the expressions of Hsp70 were increased while the expression of p53 and caspase-3 were decreased in the stem cell metabolites samples. Conclusion: These results showed that stem cell metabolites are non-toxic, do not cause a systemic immune response to surrounding tissue, and able to inhibit the occurrence of apoptosis.

Evaluation of a lymphocyte transformation test and cytokine detection assay to identify phenytoin and carbamazepine provoked DRESS or SJS/TEN in epilepsy patients

Phenytoin (PHE) and carbamazepine (CBZ) are first rank causative drugs that can induce drug rash with eosinophilia and systemic symptoms (DRESS) and Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Identification of anti-epileptic drugs as a culprit drug has been problematic; hence, in vitro tests could be promising methods to define causative drugs without clinical risk. The aim of this study is to evaluate the efficacy of lymphocyte transformation tests (LTT) and cytokine detection assays in identifying PHE and CBZ as culprit drugs. Peripheral blood mononuclear cells were collected from normal, PHE/CBZ tolerance and PHE/CBZ hypersensitivity cohorts and utilized for cell-culture assays. LTT was performed and culture supernatants were subjected to multiple cytokine detection assays. Our study showed that LTT correlated with outcomes of Naranjo's assessment with statistical significance (r = 0.614). Various sensitivities of LTT and cytokine detection assays were demonstrated. Although both assays provided excellent specificity, LTT yielded higher sensitivity as compared to those of cytokine detection assays in both DRESS and SJS/TEN. Regardless whether specificity is, this is the first report to demonstrate that IL-4 detection assay could enhance sensitivity to identify culprit drug when it was combined with LTT for SJS/TEN patients or it was combined with IL-2/IFN-γ detection assays for DRESS ones.

The patterns of in vitro cell-death and inflammatory cytokines induced by distinct BCG vaccine strains are differentially induced in human mononuclear cells

ABSTRACTTuberculosis (TB) remains among the world's leading cause of mortality. For its control, studies of TB vaccines are needed. Since live-attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently in use, studies on the protective role of BCG are required. In this study, we analyzed host cells purified directly from whole blood of human immunodeficiency virus (HIV)-negative volunteers, comprising adult healthy donors (HD) and neonates (umbilical cord bloods, UCB), with the aim to directly compare in vitro immune responses with distinct BCG strains in human mononuclear cells. The Moreau, Pasteur, and Danish BCG strains were used to infect mononuclear cells in vitro for 48 h; bacilli viability and cell-death were subsequently detected by flow cytometry. In addition, cell culture supernatants were used in cytokine detection assays. Overall, the Moreau BCG strain induced higher levels of apoptosis than the Pasteur and Danish BCG strains in both the HD and UCB groups (p-value < 0.05), and a human monocytic cell-line mirrored those cell-death patterns after BCG infection. The Moreau BCG strain, exclusively, induced Th1 cytokines at the highest levels in cells from adults (p-value < 0.05) when compared with both Pasteur and Danish BCG strains, whereas TGF-β1 levels were reduced significantly (p-value < 0.01) in the HD group when cells were infected with the Moreau BCG vaccine. As expected, eight out of 22 pro-inflammatory cytokines were secreted at significant levels (p-value < 0.05) above the baseline rates in all BCG-infected cell cultures, in the HD group only. When analyzing these results, we excluded confounding factors related to storage and viability of the BCG strains used. These findings suggest that Moreau BCG is a more potent immunostimulating agent than the Pasteur and Danish BCG strains. Clinical trials will be needed to confirm these findings.

Polydimethylsiloxane SlipChip for mammalian cell culture applications.

This paper reports a polydimethylsiloxane (PDMS) SlipChip for in vitro cell culture applications, multiple-treatment assays, cell co-cultures, and cytokine detection assays. The PDMS SlipChip is composed of two PDMS layers with microfluidic channels on each surface that are separated by a thin silicone fluid (Si-fluid) layer. The integration of Si-fluid enables the two PDMS layers to be slid to different positions; therefore, the channel patterns can be re-arranged for various applications. The SlipChip design significantly reduces the complexity of sample handling, transportation, and treatment processes. To apply the developed SlipChip for cell culture applications, human lung adenocarcinoma epithelial cells (A549) and lung fibroblasts (MRC-5) were cultured to examine the biocompatibility of the developed PDMS SlipChip. Moreover, embryonic pluripotent stem cells (ES-D3) were also cultured in the device to evaluate the retention of their stemness in the device. The experimental results show that cell morphology, viability and proliferation are not affected when the cells are cultured in the SlipChip, indicating that the device is highly compatible with mammalian cell culture. In addition, the stemness of the ES-D3 cells was highly retained after they were cultured in the device, suggesting the feasibility of using the SlipChip for stem cell research. Various cell experiments, such as simultaneous triple staining of cells and co-culture of MRC-5 with A549 cells, were also performed to demonstrate the functionalities of the PDMS SlipChip. Furthermore, we used a cytokine detection assay to evaluate the effect of endotoxin (lipopolysaccharides, LPS) treatment on the cytokine secretion of A549 cells using the SlipChip. The developed PDMS SlipChip provides a straightforward and effective platform for various on-chip in vitro cell cultures and consequent analysis, which is promising for a number of cell biology studies and biomedical applications.

Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of Schistosomiasis mansoni infection

BackgroundThe immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area.MethodsThe activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA.ResultsThe XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4+ T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8+ T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups.ConclusionsThe data indicate that SEA-stimulated CD4+ T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group.

Emergence of a Broad Repertoire of GAD65-Specific T-Cells in Type 1 Diabetes Patients with Graft Dysfunction after Allogeneic Islet Transplantation

Islet transplantation is one of the most promising therapies for type 1 diabetes (T1D). A major issue in islet transplantation is the loss of graft function at late phase. Several studies suggested the involvement of islet-specific T-cells in such islet graft dysfunction. In this study, we investigated the breadth and type of glutamic acid decarboxylase 65 (GAD65)-specific T-cells in T1D patients after allogeneic islet transplantation. Peripheral blood mononuclear cells (PBMCs) were obtained from islet-transplanted T1D patients during insulin-independent period and cultured for 7 days with pools of GAD65 overlapping peptides in the presence of IL-2. Cytokine secretion profiles of peptide-reactive T-cells were analyzed after a short-term restimulation with the same peptides by a multiplex bead-based cytokine assay and by an intracytoplasmic cytokine detection assay. Robust GAD65-specific CD4(+) and CD8(+) T-cell responses were detected in patients who eventually developed chronic graft dysfunction. Multiple GAD65 peptides were found to induce specific T-cell responses in these patients, indicating that the repertoire of GAD65-specific T-cells was broad. Furthermore, GAD65-specific CD4(+) T-cells were composed of heterogeneous populations, which differentially expressed cytokines including IFN-γ and type 2 cytokines, but not IL-10. In contrast, patients who showed only marginal GAD65-specific T-cell responses maintained substantially longer graft survival and insulin independence. In conclusion, our study suggests that the emergence of islet-specific T-cells precedes the development of chronic graft dysfunction in islet-transplanted patients. Thus, our observations support the hypothesis that these islet-specific T-cells contribute to the development of chronic islet graft dysfunction.

Immunomodulation of Human CD8+ T Cells by Thymoglobulin In Vitro

The major mechanism of Thymoglobulin in inhibiting allograft rejection involves lymphocyte depletion. However, its role in modulating CD8+ cells has remained unclear. This study evaluated the immunologic effects of Thymoglobulin on human CD8+ cells. Purified CD8+ cells were pretreated with Thymoglobulin followed by incubation for 72 hours. The culture supernates and cells were analyzed using real-time quantitative polymerase chain reaction (RT-PCR), multiplex cytokine detection assay, and flow cytometry. RT-PCR showed that Thymoglobulin-treated CD8+ cells up-regulated transcripts for CD25, CTLA-4, OX40, GITR, Foxp3, IFN-γ, interleukin (IL)-10, and IL-2 when compared with isotype control immunoglobulin (Ig). The expression for GITR and IL-2 transcripts was down-regulated at 72 hours after incubation. The release of IFN-γ, IL-10, and IL-2 from Thymoglobulin-pretreated CD8+ cells was detected using multiplex assay after 24-hour incubation, and these cytokine levels were decreased after incubation for 48 and 72 hours. Flow cytometry demonstrated up-regulation of CD69 and CD25 expression after treatment. The surface CTLA-4 and intracellular Foxp3 expression was not increased in Thymoglobulin-treated cells. Our results demonstrated that Thymoglobulin-treated CD8+ cells up-regulate CD25, and multiple costimulatory molecules at the transcriptional level. The up-regulation of transcripts of immune regulatory cytokines accompanies the release of these cytokines. The unique effects of Thymoglobulin on CD8+ cells may be an important mechanism for its action in inducing immunosuppression and immunomodulation.

Lateral flow assay for simultaneous detection of cellular- and humoral immune responses

Objective The development of a cytokine detection assay suitable for detection of multiple biomarkers for improved diagnosis of mycobacterial diseases. Design and methods A lateral flow (LF) assay to detect IL-10 was developed utilizing the up-converting phosphor (UCP) reporter-technology. The assay was evaluated using blood samples of leprosy patients. Multiplex applications were explored targeting: 1) IL-10 and IFN-γ in assay buffer; 2) IL-10 and anti-phenolic glycolipid (PGL-I) antibodies in serum from leprosy patients. Results Detection of IL-10 below the targeted level of 100 pg/mL in serum was shown. Comparison with ELISA showed a quantitative correlation with R 2 value of 0.92. Multiplexing of cytokines and simultaneous detection of cytokine and antibody was demonstrated. Conclusions The UCP-LF IL-10 assay is a user-friendly, rapid alternative for IL-10 ELISAs, suitable for multiplex detection of different cytokines and can be merged with antibody-detection assays to simultaneously detect cellular- and humoral immunity.

Alphavirus particle filovirus vaccine activation of Dendritic cells (52.7)

Abstract Filoviruses, Marburg (MARV) and Ebola (EBOV), are among the deadliest etiological agents causing hemorrhagic fevers. Although little is known about the pathogenesis or what immunological responses are required to protect against filoviruses, a promising recombinant RNA replicon vaccine has been developed consisting of an attenuated alphavirus, Venezuelan equine encephalitis virus (VEE), to express MARV and EBOV viral genes such as the virus glycoprotein (GP). While numerous in vivo studies have demonstrated the protective efficacy of filovirus GP expressing VEE replicon particles (GP-VRP) in mice, guinea pigs, and non-human primates, when challenged with homologous virus, little has been done to understand the means by which these VRP filovirus vaccines initiate and establish immunity. In this study, we sought to dissect the protective mechanism of VRPs by evaluating the GP-VRPs effects on dendritic cells (DCs) by several characterization assays. Immunofluorescence assay (IFA), flow cytometry, and cytokine detection assays were used to assess maturation, co-stimulatory molecule expression, and cytokine expression profiles of GP-VRP-treated DCs. These findings will aid in deciphering early innate immune responses initiated by GP-VRP vaccination that are necessary for eliciting protective immunity against filovirus infection.

The role of fibroblasts in neonatal intestinal inflammation (89.46)

Abstract Background: Fetal intestinal fibroblast cell lines (FIF) produce more IL-8 in response to LPS compared to non-neonatal intestinal fibroblast lines (NNIF). Necrotizing Enterocolitis (NEC) is the most common gastrointestinal emergency in neonates and occurs more frequently in premature infants than term infants. We propose that fetal fibroblasts are inherently more responsive to stress and contribute to increased inflammation. Method: Primary human FIF and NNIF cell lines were exposed to E. coli LPS for 24 hours and the amount of IL-8 in the supernatants was determined by quantitative ELISA. A membrane blotting cytokine detection assay was also used to establish the cytokine profiles in the supernatants. Supernatants were used to stimulate neutrophils and activation was measured by increased CD11b expression by flow cytometry. Results: LPS stimulation induced a cytokine profile in FIF distinct from NNIF that included MCP-1, IL-6 and GRO-alpha. The supernatants from LPS stimulated FIF also activated neutrophils significantly more than supernatants from LPS treated NNIF. Conclusions: Fetal fibroblasts are more sensitive to stimulation by bacteria endotoxin and produce cytokines that promote inflammation, including the activation of neutrophils. Lamina propria fibroblasts in the developing gut may drive inflammation and put premature infants at an increased risk for developing NEC.

Abstract C38: Immunosuppression by indoleamine 2,3-dioxygenase in mesenchymal stem cells derived from adult human tissues

Abstract Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton's jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin (PHA)-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed PHA-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, interleukin-10, transforming growth factor-β1, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ, while that of indoleamine 2,3-dioxygenase (IDO) increased. Use of an antagonist, 1-methyl-L-tryptophan, restored T-cell proliferation and confirmed an IDO contribution to IFN-γ-induced immunosuppression by MSCs. Addition of tryptophan catabolite, such as kynurenine, 3-hydroxykynurenine, or quinolinic acid, significantly decreased PHA-induced T-cell proliferation. These data indicate that IFN-γ produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation through tryptophan depletion and the local accumulation of tryptophan catabolites. Our findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C38.

Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β 1, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.

Induction of broad repertoire of melanoma associated antigen-specific CD4+ T cells by dendritic cell vaccine loaded with killed allogeneic melanoma cells in patients with metastatic melanoma

3029 Background: Tumor-specific CD4+ T cells play an important role in antitumor immunity. CD4+ T cells recognizing melanoma associated antigens can be spontaneously generated in patients with metastatic melanoma as demonstrated by the infiltration of such cells at tumor sites; however, they are often dysfunctional. We analyzed tumor-specific CD4+ T cell repertoires in the blood of metastatic melanoma patients, and examined whether dendritic cell (DC) vaccine loaded with killed allogeneic melanoma cells administered to metastatic melanoma patients can induce and/or improve melanoma-specific CD4+ T cell immunity. Methods: PBMCs were obtained from ten metastatic melanoma patients who received at least four monthly DC vaccine injections (GM-IL-4 DC vaccine loaded with killed allogeneic melanoma cells, matured with CD40L and TNFa). Melanoma-specific T cell repertoires at baseline and after vaccinations were assessed with EPIMAX approach using overlapping peptide libraries spanning MART-1, gp100, and TRP-1 antigens. The frequencies of CD4+ T cells responding to the identified peptides were assessed by intracellular cytokine detection assay with flow cytometry. Results: At baseline, four out of ten patients displayed gp100-specific CD4+ T cells which secreted IFNg and/or IL-2, while none displayed MART-1-specific CD4+ T cells. Nine gp100 CD4+ T cell epitopes were identified in total. The T cells lacked proliferative capacity (seven out of nine gp100-specific T cells) in spite of cytokine secretion in response to peptide stimulation. We found PD-1 expression on specific CD4+ T cells which lacked the proliferative capacity. Analysis of post-vaccine showed the presence of proliferative gp100-specific CD4+ T cells in four patients. One of these patients experienced a complete response and another one a long-term survival. The frequencies of specific CD4+ T cells secreting IL-2 and/or IFNg were increased at post-vaccine. Functional MART-1 and TRP-1-specific CD4+ T cells were also induced in both patients. Conclusions: DC vaccine loaded with killed tumor cells can prime broad repertoire of tumor-specific CD4+ T cells as well as revert dysfunction of pre-existing specific T cells. No significant financial relationships to disclose.

Respiratory Syncytial Virus Inhibits Granulocyte Apoptosis through a Phosphatidylinositol 3-Kinase and NF-κB-Dependent Mechanism

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-kappaB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.

Colorimetric Bio-Barcode Amplification Assay for Cytokines

The bio-barcode amplification assay has become a powerful tool in detecting tens to hundreds of biological targets such as proteins and nucleic acids in the entire sample. However, current bio-barcode detection schemes still require many experimental steps including microarrayer-based immobilization of oligonucleotides on a glass chip, silver enhancement of immobilized gold nanoparticles on a chip, and light-scattering measurement. Here, we report a colorimetric bio-barcode method that minimizes the above requirements while detecting 30 aM concentrations of cytokines (approximately 3 orders of magnitude more sensitive than conventional nonenzymatic cytokine detection assays). The assay is based on porous microparticles, which enable loading of a large number of barcode DNA per particle, and gold nanoparticle-based colorimetric barcode detection method.

Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies

We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4 + and CD8 + T cells in small volumes of whole blood from infants from developing countries. The assay is performed in two steps. First, whole blood is stimulated in the presence of specific antigens for 6–18 h, ending with cryopreservation of fixed white cells. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting. Later, in a more resourceful setting, interferon-gamma producing CD4 + or CD8 + T cells are detected by flow cytometry. The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site.