Peripheral Blood Mononuclear Cell Subsets Research Articles

The immunomodulatory effects of bevacizumab on systemic immunity in patients with metastatic melanoma

Metastatic melanoma is near-to-invariably a fatal disease. As novel therapeutic strategies against metastatic melanoma are urgently needed, we have tested a combinatorial regimen consisting of conventional chemotherapy coupled to bevacizumab, a monoclonal antibody that inhibit angiogenesis, demonstrating some clinical benefit. A preliminary assessment of one of our clinical trials points to a previously unrecognized immunomodulatory effect of bevacizumab. Herein, we evaluate the immunomodulatory effect of bevacizumab when administered together with conventional chemotherapy to patients with metastatic melanoma. To this aim, we measured the abundance of various lymphocyte subsets among peripheral blood mononuclear cells (PBMCs) as well as the circulating levels of 42 cytokines, chemokines and growth factors in patients with metastatic melanoma who received albumin-bound paclitaxel plus carboplatin, either as a standalone intervention (AC, 55 subjects) or combined with bevacizumab (ACB, 39 individuals), in the context of clinical trials N057e and N0775, respectively. Relative shifts in PBMC subsets and cytokine levels were calculated (relative to baseline levels) when patients underwent restaging evaluation after two cycles of therapy. The Mann–Whitney U test was used to compare responses between the groups. Bevacizumab failed to affect the TH1/TH2 cell ratio in this patient cohort. However, we observed a significant increase in CD8+ lymphocytes in patients who received ACB (+38%) but not in subjects treated with AC only (−10%) (p = 0.03). Moreover, circulating interleuikin-6 (IL-6) levels were reduced in patients treated with ACB (−42%) but not in individuals receiving AC only (28%) (p = 0.0018). Thus, the addition of bevacizumab to chemotherapy for the treatment of metastatic melanoma exerts immunomodulatory effects.

Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Phenotypic and functional traits of peripheral blood mononuclear cells retained by controlled cryopreservation: implications for reliable sequential studies of dynamic interactions with endothelial cells

Many immunological experiments would be greatly facilitated if peripheral blood mononuclear cells (PBMC) preserved important functional properties over longer periods of time. Regarding adhesive interactions with endothelial cells, a crucial step of inflammatory processes, this had not been investigated yet. We demonstrate that PBMC subsets subjected to controlled cryopreservation retain their phenotypic traits inasmuch as the proportion of viable T cells, monocytes/macrophages and B cells was comparable with their freshly isolated counterparts. More importantly, we demonstrate for the first time that the procedure does not impede crucial adhesion-mediated dynamic interactions with endothelial cells. Using a flow chamber system, freshly isolated and cryopreserved PBMC showed similar rolling and firm adhesion on TNF-α-activated endothelial cells under shear flow as compared to freshly isolated PBMC from the same donors. Thus, our observation is an important prerequisite for functional studies of leucocyte recruitment when sequential investigations with material from the same patients are required.

Cytokine production by non-stimulated peripheral blood NK cells and lymphocytes in early-onset severe pre-eclampsia without HELLP

Preeclampsia involves an exacerbated maternal inflammatory response that suggests a possible role of innate immunity. NK cells can promote this kind of response through cytokine production and the expression of activating or inhibitory receptors. The aims of the present study were to explore cytokine production by peripheral blood mononuclear cells, as well as cytotoxic ability and receptor expression for HLA-E and HLA-G molecules in peripheral natural killer (NK) cells of women with early-onset severe preeclampsia without HELLP (hemolysis, elevated liver enzyme levels and a low platelet count) syndrome. The expression of the ILT2, KIRDL4, NKG2A, and NKG2C receptors and of cytotoxic activity was measured in non-stimulated NK cells, whereas the intracellular expression of IL-4, IL-10, IL-13, IL-12, IFNγ, TNF and VEGF, was assessed in non-stimulated peripheral blood mononuclear cells subsets using flow cytometry. Circulating soluble HLA-G was also determined by ELISA. The intracellular cytokines tested were significantly higher in NK cell subsets from severely preeclamptic women compared with the control group. On the other hand, the percentage of NK cells expressing NKG2A or NKG2C and the cytotoxic activity of NK cells were significantly higher in severely preeclamptic women. Furthermore, there was a significant correlation between urine protein concentration and soluble human leukocyte antigen G (soluble HLA-G) in serum. We conclude that patients with early-onset severe preeclampsia without HELLP syndrome have increased NK cell function related to cytokine production, cytotoxicity and expression of lectin-like receptors such as NKG2.

AngiomiR-126 expression and secretion from circulating CD34+ and CD14+ PBMCs: role for proangiogenic effects and alterations in type 2 diabetics

AbstractSeveral peripheral blood mononuclear cell (PBMC)–derived cell populations can promote angiogenesis, and differences in CD34+ or CD14+ surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34+/CD14+, CD34+/CD14−, CD34−/CD14+, and CD34−/CD14− PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34+/14+ and CD34+/14− PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34− PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34+ compared with CD34− PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti–miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34+ PBMCs. MiR-126 levels in supernatants of CD34+ PBMC subsets were substantially higher compared with CD34− PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34+ PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34+ PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34+ PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34+ PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

Abstract B71: Protective immunizations using cultured peripheral blood cells.

Abstract Murine models of Autologous Cellular Immunotherapy (ACI) have been useful tools for understanding the mechanisms by which human immunotherapy may work. Sipuleucel-T is an FDA approved therapeutic for the treatment of asymptomatic or minimally symptomatic, metastatic castration-resistant metastatic prostate cancer; Phase 3 studies demonstrated a statistically significant prolongation in overall survival when compared to a control group. Sipuleucel-T is manufactured by activating peripheral blood mononuclear cells (PBMC), including antigen presenting cells, with a recombinant protein fusion of prostatic acid phosphatase and granulocyte macrophage colony-stimulating factor (GM-CSF). We have previously reported a method for the use of cultured PBMC in a pre-clinical setting to elicit a T cell response against a tumor associated antigen, and to protect mice from tumors expressing that antigen. The model antigen we used was human Carbonic Anhydrase IX (hCA9). Mouse PBMCs were cultured with the antigen fused to murine GM-CSF (hCA9-GM) or with GM-CSF alone. Mice were immunized 3 times with these cells at 2 week intervals. Satellite mice were then harvested for evaluation of in vitro T cell response (antigen recall assay) and the remaining mice were inoculated with hCA9-expressing tumor cells, and tumor growth followed over time. Immunized mice mounted a robust T cell response in vitro to hCA9 in a dose dependent manner, a result that correlated with protection from tumor challenge in mice immunized with PBMC cultured in hCA9-GM. Herein, we investigate the cellular requirements for protection against tumor challenge and ability of a selective Toll-like receptor (TLR) 9 agonist to enhance the potency of this cellular immunotherapy. To investigate the importance of T cells for anti-tumor responses, we demonstrate that selective depletion of either CD4 or CD8 T cells results in a diminution or loss of protection toward tumor challenge; indicating T cells are necessary in vivo to prevent tumor growth. Next, to stimulate antigen specific T cell activity in the ACI therapy, relative to hCA9 antigen alone, cells were cultured with hCA9-GM antigen in combination with the TLR9 agonist CpG1826. Addition of CpG1826 resulted in significant accumulation of pro-inflammatory cytokines (IL-1b, IL-6, IL-10, TNFa, KC and IFNg) in the culture medium, and to higher surface expression of activation and co-stimulatory markers (CD25, CD69, CD80, CD86, and MHC Class II) on cultured cells. Moreover, inclusion of CpG1826 to cultures enhanced protection against tumor cell challenge. Interestingly, this enhancement in potency was not associated with increased antigen specific T cell responses, as measured in the in vitro antigen recall assay. In conclusion, we show that, in mice, unfractionated white blood cells collected from peripheral blood can be harnessed to mount effective anti-tumor immune responses and that TLR9 agonists complement this approach. Furthermore, this active immunotherapy triggers a balanced immune response relying on the activity of both CD4 and CD8 T cells. Future studies will be aimed at evaluating the role of various PBMC subsets (T cell, B cell and myeloid cell) in protecting mice from tumor cell challenge. Citation Format: Kenneth A. Brasel, Craig Meagher, Marykay Ligocki, Lauren Cerretti, Felecia Wagener, Sam Li, James Trager. Protective immunizations using cultured peripheral blood cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B71.

Cellular sources of dysregulated cytokines in relapsing-remitting multiple sclerosis

BackgroundNumerous cytokines are implicated in the immunopathogenesis of multiple sclerosis (MS), but studies are often limited to whole blood (WB) or peripheral blood mononuclear cells (PBMCs), thereby omitting important information about the cellular origin of the cytokines. Knowledge about the relation between blood and cerebrospinal fluid (CSF) cell expression of cytokines and the cellular source of CSF cytokines is even more scarce.MethodsWe studied gene expression of a broad panel of cytokines in WB from relapsing-remitting multiple sclerosis (RRMS) patients in remission and healthy controls (HCs). Subsequently we determined the gene expression of the dysregulated cytokines in isolated PBMC subsets (CD4+, CD8+T-cells, NK-cells, B-cells, monocytes and dendritic cells) from RRMS patients and HCs and in CSF-cells from RRMS patients in clinical relapse and non-inflammatory neurological controls (NIND).ResultsRRMS patients had increased expression of IFN-gamma (IFNG), interleukin (IL) 1-beta (IL1B), IL7, IL10, IL12A, IL15, IL23, IL27, lymphotoxin-alpha (LTA) and lymphotoxin-beta (LTB) in WB. In PBMC subsets the main sources of pro-inflammatory cytokines were T- and B-cells, whereas monocytes were the most prominent source of immunoregulatory cytokines. In CSF-cells, RRMS patients had increased expression of IFNG and CD19 and decreased expression of IL10 and CD14 compared to NINDs. CD19 expression correlated with expression of IFNG, IL7, IL12A, IL15 and LTA whereas CD14 expression correlated with IL10 expression.ConclusionsUsing a systematic approach, we show that expression of pro-inflammatory cytokines in peripheral blood primarily originates from T- and B-cells, with an important exception of IFNG which is most strongly expressed by NK-cells. In CSF-cell studies, B-cells appear to be enriched in RRMS and associated with expression of pro-inflammatory cytokines; contrarily, monocytes are relatively scarce in CSF from RRMS patients and are associated with IL10 expression. Thus, our findings suggest a pathogenetic role of B-cells and an immunoregulatory role of monocytes in RRMS.

SERPINB3 expression on B-cell surface in autoimmune diseases and hepatitis C virus-related chronic liver infection

SERPINB3 is a serine protease inhibitor with pleiotropic functions. It is involved in several physiological and pathological processes, where it appears to exert antiapoptotic effects. Little is known about its expression on immune system cells, the major players in mechanisms of viral defense and autoimmune disorders. The aim of this study was to characterize the expression of SERPINB3 on the surface of peripheral blood mononuclear cell subsets in both normal subjects and in patients with chronic viral infections and autoimmune diseases. Sixty-two patients were analyzed by flow cytometric analysis, including 45 with hepatitis C virus (HCV)-related chronic liver disease and 17 with systemic lupus erythematosus (SLE). SERPINB3 was expressed on B lymphocytes in 79% of the controls, in 32% of the HCV-infected patients and in none of the SLE patients. Surface localization of SERPINB3 was confirmed by confocal microscopy. SERPINB3 positivity was associated with CD27 reactivity (r = 0.98), but not to other activation molecules (CD69, CD71, CD86 and CXCR3). SERPINB3 is physiologically expressed on the surface of CD27(+) B lymphocytes, but its expression is reduced in HCV viral infection and not detectable in SLE patients. These results may suggest a role for SERPINB3 in B-cell defects typically found in viral infections and autoimmune disorders.

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

Cellular immune response in prognosis of Bell's palsy and its relation to clinical and electrophysiological findings

ObjectiveTo determine the cellular immune response in Bell's palsy (BP) and its prognostic value in relation to clinical and electrophysiological findings. MethodsTwenty patients with BP were subjected to: Facial nerve paralysis assessment according to House–Brackmann (H&B) grading system, bilateral facial nerve conduction study with electroneurography (ENoG) quotient calculation, blink reflex, and needle electromyography (EMG) for the affected side; one week from the onset. Before the start of medical treatment, peripheral blood mononuclear cell subsets were analyzed to reveal the percentage of total T cells (CD3+), T helper/inducer cells (CD4+), T cytotoxic (CD8+) and B cells (CD19+). Patients were followed up by H&B, ENoG and needle EMG up to 3months from the onset (end point). Fifteen age and sex matched healthy control subjects for the electrophysiological study and laboratory tests were included. ResultsThe percentages of CD3+, CD4+ & CD8+ were significantly depressed in BP patients than in control. CD19+ percentage did not show significant difference between them. On follow up, H&B revealed significant improvement. Neither electrophysiological parameters nor ENoG showed significant difference between initial and follow up assessments. Initial CD4+ percentage correlated negatively with disease duration. While Initial CD8+ percentage correlated positively with follow up compound muscle action potential (CMAP) amplitude of orbicularis oris muscle and ENoG of orbicularis oculi and nasalis muscles. Initial CD19+ percentage correlated negatively with follow up H&B and R1 & R2 responses of follow up blink reflex. Initial CD3+ percentage did not correlate with any of the follow up measures. ConclusionDecreased percentage of peripheral blood CD3+, CD4+ & CD8+ in BP patients emphasizes the role of cell mediated autoimmune pathogenesis in the acute stage of the disease. These cells have a prognostic significance for prediction of the disease duration and outcome. Analysis of T lymphocytes subsets may provide an additional parameter to differentiate patients with favorable from those with poor prognosis.

Abstract 16896: Kinin-Mediated Recruitment of Circulating Endothelial Supporting Cells is Impaired in Patients with CAD or Diabetes: Impact on Endothelial Healing

Purpose: We have investigated the role of vascular-derived kinins for the recruitment of angiogenic monocytes and circulating angiogenic progenitor cells (CPC) to the vessel wall, and the functionality of this mechanism during coronary artery disease (CAD) and type 2 diabetes mellitus (T2D). Methods: Expression of the kinin B2 receptor (B2R) on peripheral blood mononuclear cell (PBMC) subsets of CAD patients with or without T2D, and age- matched healthy subjects (HS) was assessed by flow cytometry. Adhesion to an endothelial monolayer and subsequent healing of a scratch gap in the endothelium was studied in vitro. In vivo, recruitment of systemically injected human PBMC to injured carotid endothelium was assessed in a mouse model. B2R blockade by icatibant or B2R deficient bone marrow cells (B2R-/- BMC) were used in those analyses to verify the relevance of the B2R. Results: In HS PBMC, B2R was low expressed (<1000MFI) on CD14hi inflammatory monocytes and highest in angiogenic Tie2+ or KDR+ monocytes and in KDR+ or CXCR4+ angiogenic CPCs (2517 to 7516MFI; P<0.05 vs. CD14hi). Recruitment of healthy CXCR4+ CPC to the endothelium was blocked by B2R inhibition in vitro (-67% vs. vehicle, P<0.05) and in vivo (-58% vs. vehicle; P<0.05), while adhesion of CD14hi monocytes was unchanged, indicating a critical role of B2R for vascular homing of CPCs, but not of CD14hi monocytes. Kinin receptor expression on CXCR4+ PC was reduced in CAD patients with or without T2D (-75% vs. H; P<0.05). Endothelial recruitment of CXCR4+ PC from T2D patients was markedly impaired and not regulated by the B2R. In vitro, endothelial gap closure was, partially mediated by the B2R during adhesion of healthy PBMC, which was lost in patients with T2D PBMC. Adenoviral B2R overexpression increased in vitro endothelial healing, while silencing of B2R abolished the recruitment and acceleration of gap closure by healthy CPC. In vivo endothelial healing was impaired in B2R-/- derived BMC that were transplanted in mice with endothelial injury (24.7% vs. 36.1%, P<0.05). Conclusions: We newly describe that vascular kinins recruit endothelial-supportive circulating cells to the vessel wall via B2R on the circulating cell. The loss of B2R on those cells might contribute to the loss of endothelial function in T2D.

Peripheral blood immunologic phenotype of population‐based breast cancer cases and matched controls

Eur J Clin Invest 2012; 42 (5): 572–574

Cell subset prediction for blood genomic studies

BackgroundGenome-wide transcriptional profiling of patient blood samples offers a powerful tool to investigate underlying disease mechanisms and personalized treatment decisions. Most studies are based on analysis of total peripheral blood mononuclear cells (PBMCs), a mixed population. In this case, accuracy is inherently limited since cell subset-specific differential expression of gene signatures will be diluted by RNA from other cells. While using specific PBMC subsets for transcriptional profiling would improve our ability to extract knowledge from these data, it is rarely obvious which cell subset(s) will be the most informative.ResultsWe have developed a computational method (Subset Prediction from Enrichment Correlation, SPEC) to predict the cellular source for a pre-defined list of genes (i.e. a gene signature) using only data from total PBMCs. SPEC does not rely on the occurrence of cell subset-specific genes in the signature, but rather takes advantage of correlations with subset-specific genes across a set of samples. Validation using multiple experimental datasets demonstrates that SPEC can accurately identify the source of a gene signature as myeloid or lymphoid, as well as differentiate between B cells, T cells, NK cells and monocytes. Using SPEC, we predict that myeloid cells are the source of the interferon-therapy response gene signature associated with HCV patients who are non-responsive to standard therapy.ConclusionsSPEC is a powerful technique for blood genomic studies. It can help identify specific cell subsets that are important for understanding disease and therapy response. SPEC is widely applicable since only gene expression profiles from total PBMCs are required, and thus it can easily be used to mine the massive amount of existing microarray or RNA-seq data.

Single Cell Network Profiling (SCNP) of IFN-α Signaling Pathways In Peripheral Blood Mononuclear Cells From Healthy Donors: Implications for Disease Characterization, Treatment Selection, and Drug Discovery

AbstractAbstract 4867 Background:The antiviral and antitumor effects of IFN-α, have been exploited for the treatment of viral infections such as hepatitis C (HCV) as well as for various malignancies, such as hairy cell leukemia and melanoma. However, widespread use of IFN-α for these and other indications is severely hampered by significant side effects which can have a major impact on patient quality of life. Thus, a greater understanding of intracellular signaling pathways regulated by IFN-α may guide in the selection of patients whose disease will have an optimal response with tolerable side effects to this cytokine. Specifically, the Signal Transducer and Activation of Transcription (Stat) transcription factors are known to play a critical role in transducing IFN-α mediated signals. Single cell network profiling (SCNP) is a multiparameter flow-cytometry based approach that can be used to simultaneously measure extracellular surface makers and intracellular signaling proteins in individual cells in response to externally added modulators. Here, we use SCNP to interrogate IFN-α signaling pathways in multiple cell subsets within peripheral blood mononuclear cells (PBMCs) from healthy donors. Objectives:This study was designed to apply SCNP to generate a map of IFN-α-mediated signaling responses, with emphasis on Stat proteins, in PBMCs from healthy donors. The data provides a reference for future studies using PBMCs from patient samples in which IFN-α-mediated signaling is aberrantly regulated. Methods:IFN-α-mediated signaling responses were measured by SCNP in PBMC samples from 12 healthy donors. PBMCs were processed for flow cytometry by fixation and permeabilization followed by incubation with fluorochrome-conjugated antibodies that recognize extracellular lineage markers and intracellular signaling molecules. The levels of several phospho-proteins (p-Stat1, p-Stat3, p-Stat4, p-Stat5, p-Stat6, and p-p38) were measured in multiple cell populations (CD14+ monocytes, CD20+ B cells, CD4+ CD3+ T cells, and CD4- CD3+ T cells) at 15 minutes, 1, 2 and 4 hours post IFN-α exposure. Results:The data revealed distinct phospho-protein activation patterns in different cell subsets within PBMCs in response to IFN-α exposure. For example, activation of p-Stat4 was detected in T cell subsets (both CD4+ and CD4- T cells), but not in monocytes or B cells. Such cell-type specific activation patterns likely play a key role in mediating specific functions within different cell types in response to IFN-α. Differences in the kinetics of activation by IFN-α for different phospho-proteins were also observed. The peak response for activation of p-Stat1, p-Stat3, and p-Stat5 was at 15 minutes in most of the cell types interrogated in this study, whereas for the activation of p-Stat4, p-Stat6, and p-p38 it was at 1 hr in the majority of cell types tested. The relationships between phospho-protein readouts in each cell subset were determined by calculating the Pearson correlation coefficients. For example, the activation of p-Stat1 and p-Stat5 at 15 minutes was positively correlated in both B cells and T cells. Conclusions:In this study, we have measured the activation of intracellular signaling proteins with emphasis on Stat transcription factors in PBMC subsets from healthy donors. We have analyzed the relationships between the activation states of phospho-proteins in the IFN-α signaling network. Characterization of IFN-α signaling pathways in samples from healthy donors has provided a network map that can be used as a reference for identifying alterations in IFN-α signaling that are the consequence of disease and/or therapeutic intervention. Future studies using SCNP to characterize IFN-α signaling pathways in PBMCs from patients with diseases such as viral infections or cancer may enable the optimization of IFN-α dosing and the identification of patient stratification biomarkers as well as the discovery of novel therapeutic agents. Disclosures:Longo: Nodality: Employment, Equity Ownership. Evensen: Nodality: Employment, Equity Ownership. Fantl: Nodality: Equity Ownership. Cesano: Nodality Inc.: Employment, Equity Ownership.

Lenalidomide Enhances Multiple Myeloma Cytotoxicity Induced by a Novel Fc Domain-Engineered Anti-HM1.24 Monoclonal Antibody with Augmented NK Cell Degranulation

AbstractAbstract 4064HM1.24, an immunological target highly expressed on majority of multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). Recently, XmAb5592, a novel Fc-domain engineered humanized anti-HM1.24 mAb with specific Fc-domain modification, was shown to induce >10-fold antibody-dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis against MM cells, when compared with the humanized normal anti-HM1.24 IgG1 (XmAb5627) from which it is derived (ASH Abstract#609, 2009). Here we investigated whether XmAb5592, when combined with other anti-MM drugs, further enhanced ADCC against MM cell lines and primary patient MM cells using Calcein-AM release ADCC assays and flow cytometric analysis for cell membrane CD107a to specifically quantitate NK cell (CD56+CD3-) activation. Addition of lenalidomide (10 μ M) in standard ADCC assays increased XmAb5592 (0.1 μ g/ml)-induced cell lysis against MM1S, MM1R, and RPMI8226 cells in the presence of peripheral blood mononuclear cells (PBMCs) from normal donors (n=2). Specifically, XmAb5592 (0.01, and 0.1 μ g/ml) combined with lenalidomide (2.5, 5, 10 μ M), in the presence or absence of IL-2 (100 units/ml), synergistically induced NK-mediated RPMI8226 MM cell lysis, as evidenced by combination index (CI) < 1 (0.08-0.89). Pre-incubation of PBMCs with additional IL-2 (100 units/ml) enhanced even stronger XmAb5592-induced cytotoxicity against MM cells than pretreatment with lenalidomide alone. Lenalidomide also enhanced PBMC effectors to kill primary patient MM cells. Cell surface CD107a, as a functional marker for NK cell activation dependent on granzyme B secretion, was further determined following target myeloma cell incubation, with or without NK cells and in the presence of mAbs. NK cells were activated by as low as 0.001 μ g/ml of XmAb5592 only in the presence of MM1S tumor cells. In contrast, neither XmAb6166, an Fc-domain knockout of XmAb5592 without NK binding, nor XmAb4614, a similar Fc-engineered mAb targeting respiratory syncytial virus (RSV) antigen, induced any cell surface CD107a on NK cells incubated with MM cells. Specific XmAb5592-induced CD107a-dependent NK cell degranulation further correlated with IFNγ secretion (r=0.7, p=0.03). XmAb5592 induced > 10-fold more potent NK degranulation than XmAb5627, which significantly correlated with MM cell lysis by ADCC, even in the presence of bone marrow stromal cells (BMSCs). Moreover, no significant HM1.24 was expressed on NK cells stimulated with or without IFN-α, suggesting minimal NK toxicity. XmAb5592 more potently (>10-fold) than XmAb5627 induces homotypic aggregation and adhesion of NK cells, which was further enhanced by lenalidomide. Finally, minimal HM1.24 expression was confirmed on different PBMC subsets including CD15+ (PMN), CD19+ (B), CD14+ (MC), and CD3+ (T) cells. These results indicate that lenalidomide further potentiates XmAb5592-induced myeloma cell killing via NK-mediated ADCC, providing a rationale to combine both novel drugs to improve patient outcome in MM. Disclosures:Muchhal:Xencor Inc: Employment. Desjarlais:Xencor Inc: Employment. Richardson:Gentium: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Heme oxygenase-1 is induced in peripheral blood mononuclear cells of patients with acute pancreatitis: a potential therapeutic target

Heme oxygenase-1 (HO-1) induction by hemin or Panhematin protects against experimental pancreatitis. As a preclinical first step toward determining whether HO-1 upregulation is a viable target in acute pancreatitis (AP) patients, we tested the hypothesis that HO-1 expression in peripheral blood mononuclear cell (PBMC) subsets of hospitalized patients with mild AP is upregulated then normalizes upon recovery and that cells from AP patients have the potential to upregulate their HO-1 ex vivo if exposed to Panhematin. PBMCs were isolated on days 1 and 3 of hospitalization from the blood of 18 AP patients, and PMBC HO-1 levels were compared with PMBCs of 15 hospitalized controls (HC) and 7 volunteer healthy controls (VC). On day 1 of hospitalization, AP patients compared with VCs had higher HO-1 expression in monocytes and neutrophils. Notably, AP monocyte HO-1 levels decreased significantly upon recovery. Panhematin induced HO-1 in ex vivo cultured AP PBMCs more readily than in HC or VC PBMCs. Furthermore, PBMCs from acutely ill AP patients on day 1 were more responsive to HO-1 induction compared with day 3 upon recovery. Similarly, mouse splenocytes had enhanced HO-1 inducibility as their pancreatitis progressed from mild to severe. In conclusion, AP leads to reversible PBMC HO-1 upregulation that is associated with clinical improvement and involves primarily monocytes. Leukocytes from AP patients or mice with AP are primed for HO-1 induction by Panhematin, which suggests that Panhematin could offer a therapeutic benefit.

Adhesion molecules are promising candidates to establish surrogate markers for natalizumab treatment

Background: Natalizumab is the first monoclonal antibody therapy approved for multiple sclerosis (MS). Its therapeutic mechanism is the blockade of the α4-integrin subunit of the adhesion molecule (AM) very late activation antigen-4 (VLA-4), which leads to an inhibition of immune cell extravasation into the central nervous system (CNS). Methods: We investigated changes in the expression levels of unblocked α4-integrin and further AM (intercellular adhesion molecule-1, -2, -3 (cICAM-1, -2, -3), leukocyte function associated antigen-1 (LFA-1)) on peripheral blood mononuclear cells (PBMC) determined by flow cytometry from 25 patients with MS before the first natalizumab infusion and before the fourth infusion. In 15 MS patients AM expression was evaluated every 3 months over 1 year. Results:We found a significant decrease (p < 0.0001) of unblocked α4-integrin cell surface expression on all investigated PBMC subsets (T cells −61.7%, B cells −69.1%, monocytes/macrophages −46.4%) in the blood of MS patients after 3 months of natalizumab treatment. Moreover, a continuous decrease (p < 0.05) of unblocked α4-integrin expression levels was seen after 3, 6, 9, and 12 months. As a secondary effect, expression levels of the other investigated AM were differentially affected. Conclusions:Results show a sustained decrease of unblocked α4-integrin expression not only in all patients but also in all investigated PBMC subsets. This probably results in a continuously decreasing transmigration of PBMC into the CNS and may explain the improved clinical efficacy in the second treatment year and also the increasing risk of progressive multifocal leukoencephalopathy during long-term natalizumab therapy. We conclude that AM expression profiles are promising candidates for the development of a biomarker system to determine both natalizumab treatment response and patients at risk for opportunistic CNS infections.

Tumor Necrosis Factor Receptor 1 Expression Is Upregulated in Dendritic Cells in Patients with Chronic HCV Who Respond to Therapy

The present studies assessed the level of tumor necrosis factor receptor (TNFR) expression in peripheral blood mononuclear cells (PBMCs) subsets from patients with chronic HCV undergoing interferon α/ribavirin-based therapy (Ifn/R). Methods. TNFR family member mRNA expression was determined using quantitative real-time PCR assays (RTPCRs) in PBMC from 39 HCV+ patients and 21 control HCV− patients. Further subset analysis of HCV + patients (untreated (U), sustained virological responders (SVR), and nonresponders (NR)/relapsers (Rel)) PBMC was performed via staining with anti-CD123, anti-CD33, anti-TNFR1 or via RTPCR for TNFR1 mRNA. Results. A similar level of TNFR1 mRNA in PBMC from untreated HCV+ genotype 1 patients and controls was noted. TNFR1 and TNFR2 mRNA levels in PBMC from HCV+ patients with SVR were statistically different than levels in HCV(−) patients. A significant difference was noted between the peak values of TNFR1 of the CD123+ PBMC isolated from SVR and the NR/Rel. Conclusion. Upregulation of TNFR1 expression, occurring in a specific subset of CD123+ dendritic cells, appeared in HCV+ patients with SVR.

Altered Expression of TNF-α Signaling Pathway Proteins in Systemic Lupus Erythematosus

To investigate the expression of tumor necrosis factor receptors (TNFR1 and TNFR2) and adapter proteins (TRADD, RIP, and TRAF2) in peripheral blood mononuclear cell (PBMC) subsets from patients with systemic lupus erythematosus (SLE). PBMC were isolated from 45 SLE patients and 25 controls, and stained with labeled antibodies that enabled identification of various T cell, B cell, and monocyte subpopulations. Expression of TNF-related signaling molecules was measured by staining with labeled antibodies either directly or following fixation and permeabilization. Apoptosis was quantified using an anti-active caspase 3 antibody. RNA expression of TNF-related signaling molecules was assessed by quantitative RT-PCR and serum levels of TNF-alpha by ELISA. SLE patients had increased levels of TNFR1, TNFR2, and TRAF2, together with decreased levels of RIP, on various B, CD4+ T, and CD8+ T cell subsets as compared to controls. This altered expression was seen in both naive and memory subpopulations, and reflected altered staining of the whole population rather than a subset of cells that were activated. The levels of these molecules were not significantly correlated with serum TNF-alpha levels or their RNA expression in whole peripheral blood. TNFR1 and TNFR2 expression was negatively correlated with disease activity. There was no association between the proportion of apoptotic cells in any of the subpopulations and serum TNF-alpha levels or expression of TNF-related signaling molecules. Patients with SLE had altered expression of TNF-related signaling molecules, suggesting that there may be an imbalance in TNF-alpha signaling favoring cellular activation as opposed to proapoptotic pathways.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 10 5, 10 6 and 10 7 bacteria/mL were co-cultured with PBMC (10 6/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56 +), total T cells (CD3 +), cytotoxic T cells (CD8 +) and CD4 + T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8 + NK subset was preferentially induced to highly express CD69 (∼90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8 + T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4 + T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria ( p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.