Lymphocyte Chemokine Research Articles

Dynamic Patterns of Clonal Evolution in Tumor Vasculature Underlie Alterations in Lymphocyte-Endothelial Recognition to Foster Tumor Immune Escape.

Although tumor blood vessels have been a major therapeutic target for cancer chemotherapy, little is known regarding the stepwise development of the tumor microenvironment. Here, we use a multicolor Cre-dependent marker system to trace clonality within the tumor microenvironment to show that tumor blood vessels follow a pattern of dynamic clonal evolution. In an advanced melanoma tumor microenvironment, the vast majority of tumor vasculature clones are derived from a common precursor. Quantitative lineage analysis reveals founder clones diminish in frequency and are replaced by subclones as tumors evolve. These tumor-specific blood vessels are characterized by a developmental switch to a more invasive and immunologically silent phenotype. Gene expression profiling and pathway analysis reveals selection for traits promoting upregulation of alternative angiogenic programs such as unregulated HGF-MET signaling and enhanced autocrine signaling through VEGF and PDGF. Furthermore, we show a developmental switch in the expression of functionally significant primary lymphocyte adhesion molecules on tumor endothelium, such as the loss in expression of the mucosal addressin MAdCAM-1, whose counter receptor a4β7 on lymphocytes controls lymphocyte homing. Changes in adhesive properties on tumor endothelial subclones are accompanied by decreases in expression of lymphocyte chemokines CXCL16, CXCL13, CXCL12, CXCL9, CXCL10, and CCL19. These evolutionary patterns in the expressed genetic program within tumor endothelium will have both a quantitative and functional impact on lymphocyte distribution that may well influence tumor immune function and underlie escape mechanisms from current antiangiogenic pharmacotherapies.

Does peripheral blood NK cell associate with hyperglycemic disorders in pregnant women?

Immune gender differences have been reported, but are little studied in elderly humans. We compared monocyte and lymphocyte subsets, along with soluble immune mediators in healthy men and women over the age of 70. We also measured natural killer (NK) lymphocyte cytotoxic granule exocytosis, chemokine synthesis, and cytokine synthesis in response to a variety of stimuli. Elderly women had significantly more circulating B cells than men, whereas men had more CD4 central memory T cells and higher monocyte levels. Plasma adiponectin levels were higher in women, plasma retinol-binding protein 4 levels were higher in men, but there were no significant gender differences in C-reactive protein, IL-15, or sphingosine-1-phosphate. Women had a higher ratio of immature CD56bright NK cells to mature CD56dim NK cells, indicating a gender difference in NK cell maturation in the elderly. Comparing sexes, female mature NK cells had more vigorous cytotoxic granule responses to K562 leukemia cells and IFN-γ responses to NKp46 crosslinking. Moreover, female NK cells were more likely to produce MIP-1β in response to a variety of stimuli. These data show that gender influences NK cell activity in elderly humans.

Advances in Research on the Role of Chemokines in Occurrence and Development of Autoimmune Thyroid Disease

AbstractChemokines can be divided into four categories: α, β, γ, and δ. Chemokine α is related to neutrophil chemotaxis. Chemokine β is correlated with adsorption of monocytes, basophils, and eosinophils. Chemokine γ is mainly a lymphocyte chemokine. Function of chemokine δ remains unclear. Chemokines α and β are primarily related to occurrence and development of autoimmune thyroid disease. This study reviews chemokines and their receptors that are related to Graves’ disease and Hashimoto’s thyroiditis.

Expression of the Lymphocyte Chemokine XCL1 in Lung Tissue of COPD Mice, and Its Relationship to CD4(+)/CD8(+) Ratio and IL-2.

We tested the role of the lymphocyte chemokine XCL1 in a murine model of chronic obstructive pulmonary disease (COPD). COPD was instigated in mice by a 12-week exposure to cigarette smoke. Some COPD animals additionally received intranasal injections of recombinant XCL1. We demonstrated that the expression of CD4(+) T cells diminished, while CD8(+) counts increased in the lungs of COPD mice, and that these changes were further aggravated by exogenous XCL1. The levels of XCL1 negatively correlated with CD4(+)/CD8(+) ratio (r=-0.945, p<0.05). In addition, the levels of this chemokine negatively correlated with the levels of interleukin-2, the lymphokine that is produced by activated CD4(+). This observation further supported negative association between XCL1 and CD4(+) response in an experimental model of COPD. In conclusion, murine model of COPD is characterized by elevated levels of XCL1, increased counts of CD8(+) T cells and diminished ratio of CD4(+)/CD8(+) cells, and decreased activity of CD4(+) T cells. This indicates that XCL1 is involved in chronic inflammatory processes in COPD and may be an important pharmacological target in this disease.

Differential modulation of immune response and cytokine profiles in the bursae and spleen of chickens infected with very virulent infectious bursal disease virus.

BackgroundVery virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius.ResultsThe viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels.ConclusionThis study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi.

Exercise training, lymphocyte subsets and their cytokines production: experience of an Italian professional football team and their impact on allergy.

Background. In recent years, numerous articles have attempted to shed light on our understanding of the pathophysiological mechanisms of exercise-induced immunologic changes and their impact on allergy and asthma. It is known that lymphocyte subclasses, cytokines, and chemokines show modifications after exercise, but outcomes can be affected by the type of exercise as well as by its intensity and duration. Interesting data have been presented in many recent studies on mouse models, but few studies on humans have been performed to check the long-term effects of exercise over a whole championship season. Methods. This study evaluated lymphocyte subsets and their intracellular IL-2, IL-4, TNF-α, and IFN-γ production in professional football (soccer) players, at three stages of the season, to evaluate if alterations occur, particularly in relation to their allergic status. Results and Conclusion. Despite significant mid-season alterations, no significant lymphocyte subclasses count modifications, except for NKs that were significantly higher, were observed at the end. IL-2 and IL-4 producing cells showed a significant decrease (P = 0.018 and P = 0.001, but in a steady fashion for IL-4), confirming the murine data about the potential beneficial effects of aerobic exercise for allergic asthma.

Specific Associations Between Clinical Signs, Immune Cells, Disease Genetic Background and Burden in a Group of Patients with B-Cell Chronic Lymphocytic Leukemia

Traffic of tumorand normal cells through the peripheral blood (PB) of patients with B-cell chronic lymphocytic leukemia (B-CLL) to the lymph nodes (LN) or spleen/ liver sites is governed by specific changes in surface and intracellular molecule expression. The study aims to investigate the potential association between different lymphocyte subsets, chemokine receptors or genetic alterations and specific clinical signs in a group of B-CLL patients. Forty-three patients were included in the study. The expression of CCR7, CXCR5, CXCR3, CCR4, CD3, CD4, CD8, CD27, CD28, CD45RA, CD25, CD127, CD38 was tested by multiparameter flow cytometry. Genetic alterations were determined by MLPA. We found increased frequency of CD38+ B-CLL cells directly correlated with the presence of LN>5cm. CXCR5 and CCR7 are homogenously expressed by monoclonal B-CLL cells. CCR4+ B-CLL cell frequency is found to be lower in the PB of patients presenting particular LN involvement. Heterogeneous and complex genetic alterations were found and only the presence of trisomy 12 associated with less frequent axillary LN involvement. We also report a significant increase in the frequency of total T cells and T cell subsets (effectorand central memory CD4+ T cells, regulatory T cells, follicular T helper cells, distinct functional CD8+ T cells) with the occurrence of specific clinical manifestations. Chemokine receptor expression on circulating CD4+ T cell subsets was augmented in connection to some specific LN locations. Consequently, clinical manifestations in B-CLL are linked to both, factors intrinsic to the monoclonal B cells, and external influences coming from the microenvironment.

Alteration in lymphocyte responses, cytokine and chemokine profiles in chickens infected with genotype VII and VIII velogenic Newcastle disease virus

Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus-host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM+ B cells and KUL01+ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P<0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P<0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions.

Manufacturing porcine islets: culture at 22 °C has no advantage above culture at 37 °C: a gene expression evaluation

The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37°C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Porcine islets were isolated from three donors and cultured at 37°C for 1day, and then under three different conditions: 37°C for 6days (condition A); 22°C for 6days (condition B); or 22°C for 5days followed by 37°C for 1day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24h at 37°C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22°C to those in islets cultured at 37°C. Results were consistent among the preparations from the three donors. Culture of porcine islets at 37°C provides benefits over culture at 22°C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress.

Enterovirus 71 infection increases expression of interferon-gamma-inducible protein 10 which protects mice by reducing viral burden in multiple tissues

Enterovirus 71 (EV71) infection has induced fatal encephalitis in thousands of young children in the Asia-Pacific region over the last decade. EV71 infection continues to cause serious problems in areas with outbreaks, because vaccines and antiviral therapies are not available. Lymphocytes are present in the brains of infected patients and mice, and they protect mice from infection by decreasing the viral burden. The chemokines responsible for recruiting lymphocytes to infected organs are yet to be identified. Among the lymphocyte chemokines detected, high levels of interferon-gamma-inducible protein-10 (IP-10) are found in the plasma and cerebral spinal fluid of patients with brainstem encephalitis as compared with the levels of a monokine induced by gamma interferon (Mig). Using a murine model to investigate the induction of IP-10 by EV71 infection, we observed that EV71 infection significantly enhanced IP-10 protein expression in the serum and brain, with kinetics similar to viral titres in the blood and brain. Brain neurons of infected mice expressed IP-10. Using wild-type mice and IP-10 gene knockout mice to investigate the role of IP-10 in EV71 infection, we found that IP-10 deficiency significantly reduced levels of Mig in serum, and levels of gamma interferon and the number of CD8 T cells in the mouse brain. Absence of IP-10 significantly increased the mortality of infected mice by 45%, with slow virus clearance in several vital tissues. Our observations are consistent with a model where EV71 infection boosts IP-10 expression to increase gamma interferon and Mig levels, infiltration of CD8 T cells, virus clearance in tissues and the survival of mice.

Effect of simvastatin on development of obliterative airway disease: An experimental study

Obliterative bronchiolitis after lung transplantation is characterized by airway inflammation leading to obliteration of small airways. Statins are known to have lipid-independent immunomodulatory properties. We investigated the effect of simvastatin treatment on innate and adaptive immune responses and the development of obliterative airway disease (OAD). In fully MHC-mismatched rat tracheal allograft recipients, we used simvastatin at different doses (0.1 to 20 mg/kg/day orally) to assess its effect on OAD development. No immunosuppressive treatment was administered. Histologic, immunohistochemical and real-time RT-PCR analyses were performed 3, 10 and 30 days after transplantation. Simvastatin treatment with doses ranging from 0.5 to 20 mg/kg/day significantly enhanced early epithelial recovery and reduced the development of OAD. No dose response was observed. Simvastatin treatment markedly reduced IL-23 mRNA and lymphocyte chemokine CCL20 production, and the infiltration of CD4(+) and CD8(+) T cells into allografts already at 3 days. At 10 days, simvastatin significantly attenuated the production of pro-inflammatory cytokines, IL-1β, TNF-α, MCP-1 and IP-10, and Th17-polarizing cytokines, IL-6 and IL-17e, and inhibited allograft infiltration by inflammatory cells. The protective effects of simvastatin on inflammation and OAD were partially mediated through nitric oxide synthase. Simvastatin treatment inhibited adaptive T-cell alloimmune activation as depicted by reduced expression of lymphocyte chemokine and pro-inflammatory cytokine mRNA and reduced allograft infiltration by inflammatory cells. Importantly, simvastatin inhibits the development of OAD and this effect is partially mediated by increased nitric oxide activity. These results suggest a role for simvastatin in the prevention of obliterative bronchiolitis.

The Inflammatory Preatherosclerotic Remodeling Induced by Intermittent Hypoxia Is Attenuated by RANTES/CCL5 Inhibition

The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients. To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA. We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure. IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia. Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.

Cross-regulation of innate response in human PBMC by an enveloped RNA virus and LPS (67.11)

Abstract The innate response to polymicrobial infections and their pathways are poorly understood. In this work, we used Sindbis virus (SIN), an enveloped RNA virus and LPS as inducers of innate response. PBMC from healthy volunteers were exposed to SIN Toto 1101, in the presence and absence of E.coli LPS. We observed absence of new virus release, SIN/GFP reporter expression and viral RNA synthesis suggesting poor replication of SIN in human PBMC. However, significant lymphocyte activation, cytokine and chemokine release were seen upon exposure of PBMC to SIN. Analysis of lymphocyte activation markers showed that, LPS was a better inducer of CD69 expression in CD4, CD8, and CD19 lymphocytes than SIN. Increased secretion of IL-12p40, IL-1β, TNF-α, IL-6, IL-18, IL-10, IL-8, RANTES, MCP-1, MIP1-α and MIP1-β was induced by LPS whereas, SIN induced IL-6, IFN-α, MCP-1, MIP1-α, MIP1-β, I-TAC and IP-10 secretion. SIN downregulated LPS induced IL-12p40 secretion, whereas LPS inhibited virus induced IFN-α and IP-10. SIN also suppressed both the spontaneous and LPS induced secretion of IFN-α, IL-17, IL-13 and SLC from PBMC. To analyze involvement of specific PBMC subsets and associated signaling events, ELISPOT and phosphoflow are used. The absence and presence of antagonism between SIN and LPS for lymphocyte activation and secretion of select cytokines and chemokines may help in understanding pathogenesis of mixed infections, and find applications in vaccine development and virotherapy.

Induction of injury to endothelium of pulmonary artery due to entero superantigen by up regulation of lymphocyte chemokine receptor 5

To observe the injurious effect of T cell activated by Staphylococcus enterotoxin B (SEB) on human pulmonary artery endothelial cell (HPAEC) and explore its possible mechanism. HPAEC was cocultured with SEB-activated T cells supernatant, and the secretion of chemotactic factors from HPAEC was examined. The Transwell inserts was used in chemoattraction assays. After HPAECs were cocultured with T cells and 10 ng/ml SEB for 3 days, HPAEC damage was monitored by microscopy and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. Three kinds of tested chemokines showed a time dependent increase in all supernatant of HPAEC incubated with different concentrations of T cells. After 72 hours, the monocyte chemoattractant protein 1 (MCP 1, ng/ml) in 1×10(-2) , 1×10(-1), 1×10(0) T cell supernatant groups was 1.240±0.103, 4.200±0.305, 6.500±0.500, respectively, macrophage inflammatory protein 1α (MIP 1α, ng/ml) was 0.210±0.015, 0.287±0.012, 0.531±0.037, respectively , and Rantes (ng/ml) was 1.420±0.074, 7.634±0.630, 15.700±1.300, respectively. Rantes presented a two phase secretion mode: in early 6 hours it increased swiftly, but relatively slow at 12, 24, 48, 72 hours. T cell adherent to polycarbonate membrane increased after SEB stimulation in superantigen group compared with control group without SEB stimulation (86.38±14.50 vs. 16.50±2.50, P<0.01). When 10 ng/ml SEB-activated T cell was cocultured with HPAEC, more of originally suspended cultured T cells adhered to HPAEC monolayer [(15.50±1.08)% vs. (1.60±0.22)%, PP<0.01], whereas the cell adhesion ratio decreased markedly in 1 μg/ml Met Rantes group [(4.39±0.66)%, PP<0.01). FACs test of HPAEC adherent T cell showed lymphocyte chemokine receptor 5 (CCR5)/CD4 and CCR5/CD8 increased over 2.5 folds and 2.8 folds compared with 100 ng/ml SEB-activated T cell. Cell death rate of HPAEC was increased when cocultured with SEB-activated T cell in superantigen group compared with HPAEC normal incubation group [(32.50±4.50)% vs. (3.50±0.50)%, P<0.01]. Increased chemoattraction and adherence of SEB-activated T cells to HPAEC could damage HPAEC; this effect was possibly due to up regulation of CCR5 on T cell.

Changes in helper lymphocyte chemokine receptor expression and elevation of IP‐10 during acute respiratory syncytial virus infection in infants

It is known that lymphopenia caused by apoptosis may occur during severe respiratory syncytial virus (RSV) infection. However, further evidence about how T-cell subsets may be affected in infants during severe RSV bronchiolitis is needed to understand the mechanisms through which immunological memory may be altered. There is increasingly convincing evidence that RSV may be associated with the development of atopy and asthma. Surrogates of Th1, Th2 and regulatory T-lymphocyte populations were measured in blood from children with acute RSV bronchiolitis and in convalescence using the cell surface receptors CXCR3, CCR4 and CD25, respectively. Samples were also obtained from healthy age-matched controls. Plasma levels of the chemokines interferon-γ inducible protein-10 (IP-10) and thymus and activation-regulated chemokine (TARC), which are known ligands for CXCR3 and CCR4, were also measured. Free plasma DNA was measured using quantitative PCR. CXCR3-positive cells were significantly decreased during acute infection (p = 0.013), while CCR4 and CD25 T-cell populations were unchanged. Plasma levels of IP-10 were markedly elevated in acute infection (p = 0.001). Convalescent samples were not significantly different to control samples for lymphocyte phenotypes or plasma chemokines. Elevated free plasma DNA was detected during acute infection compared with convalescence and controls. A profound reduction in the Th1, but not Th2, and CD25-positive lymphocyte populations associated with exaggerated IP-10 production occurs in severe RSV bronchiolitis. Free DNA is detectable in plasma. This may allow significant alterations in the generation of T-cell memory.

Effect of Elotuzumab on Circulating Lymphocytes, Chemokines, and Cytokines In Multiple Myeloma Patients

AbstractAbstract 4070Background: Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein which is highly and uniformly expressed on malignant plasma cells in multiple myeloma (MM). CS1 is also expressed at a lower level on the cell surface of natural killer (NK) cells, natural killer T-cells (NKT), and on a subset of CD8 positive T-cells, but not resting B cells, monocytes, or CD4 positive T-cells. Preclinical studies have previously demonstrated that elotuzumab kills myeloma cells via NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, in whole blood assays elotuzumab treatment resulted in the elevation of chemokines and cytokines in culture supernatants. Elotuzumab is being studied in three phase 1 clinical trials in relapsed and/or refractory MM: 1) a monotherapy dose-escalation study, 2) a combination study with bortezomib, and 3) a combination study with lenalidomide and low-dose dexamethasone. Given that NK cells express CS1, there was a concern that elotuzumab treatment could potentially lead to NK cell depletion. Therefore, the pharmacodynamic goals of these clinical studies were to examine the effects of elotuzumab treatment on lymphocyte counts (in particular NK cells), chemokine levels, and cytokine levels. Methods: Absolute lymphocyte counts were determined in peripheral blood samples using the TruCOUNT™ flow cytometry assay. Serum levels of chemokines and cytokines were measured using a multiplex, bead-based assay (Luminex®). Results: In all 3 studies, we observed no depletion of total lymphocytes or lymphocyte subsets, including CS1 positive NK cells, with elotuzumab dosing either alone or in combination with bortezomib or lenalidomide/dexamethasone. A transient decrease in the absolute number of circulating total lymphocytes (approximately 75%–90% reduction from baseline) upon first elotuzumab dose was observed, followed by a recovery of these lymphocyte counts to baseline or near baseline levels as dosing cycles continued. The transient decrease in lymphocytes included both CS1 positive and CS1 negative cell subsets. This transient decrease in lymphocyte counts was associated with increased levels of circulating chemokines and cytokines following dosing. Post-dose serum samples from study subjects had a median 16-fold (range 1.3–270-fold) elevated levels of interferon inducible protein 10 (IP-10), a chemokine well known to induce lymphocyte trafficking. Other serum analytes were elevated following the initial elotuzumab dosing, including MCP-1, IL-6, and TNF-α, also known for their role in chemotaxis and inflammatory processes. The elevated levels of chemokines and cytokines were generally not seen at subsequent doses. Conclusions: A transient decrease in both CS1 positive and CS1 negative lymphocyte counts was observed following the first dose of elotuzumab in all phase 1 studies in patients with relapsed and/or refractory MM, which resolved during subsequent dosing. This transient decrease appears to be due to lymphocyte trafficking resulting from release of chemokines and cytokines. No evidence of elotuzumab-mediated depletion of CS1 positive lymphocytes was observed. Elotuzumab treatment thus does not appear to be associated with NK cell depletion. Disclosures:Neyer:Facet Biotech: Employment. Ding:Facet Biotech: Employment. Chen:Facet Biotech: Employment. Sheridan:Facet Biotech: Employment. Rice:Facet Biotech: Employment. Balasa:Facet Biotech: Employment. Keller:Facet Biotech: Employment. Fang:Facet Biotech: Employment. Albano:Facet Biotech: Employment. Tran:Facet Biotech: Employment. Zhao:Facet Biotech: Employment. Afar:Facet Biotech: Employment.

Co-ordinated Gene Expression in the Liver and Spleen during Schistosoma japonicum Infection Regulates Cell Migration

Determining the molecular events induced in the spleen during schistosome infection is an essential step in better understanding the immunopathogenesis of schistosomiasis and the mechanisms by which schistosomes modulate the host immune response. The present study defines the transcriptional and cellular events occurring in the murine spleen during the progression of Schistosoma japonicum infection. Additionally, we compared and contrasted these results with those we have previously reported for the liver. Microarray analysis combined with flow cytometry and histochemistry demonstrated that transcriptional changes occurring in the spleen were closely related to changes in cellular composition. Additionally, the presence of alternatively activated macrophages, as indicated by up-regulation of Chi3l3 and Chi3l4 and expansion of F4/80+ macrophages, together with enhanced expression of the immunoregulatory genes ANXA1 and CAMP suggests the spleen may be an important site for the control of S. japonicum-induced immune responses. The most striking difference between the transcriptional profiles of the infected liver and spleen was the contrasting expression of chemokines and cell adhesion molecules. Lymphocyte chemokines, including the homeostatic chemokines CXCL13, CCL19 and CCL21, were significantly down-regulated in the spleen but up-regulated in the liver. Eosinophil (CCL11, CCL24), neutrophil (CXCL1) and monocyte (CXCL14, CCL12) chemokines and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver but unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Co-ordinated expression of these genes probably contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver, thereby facilitating the development of hepatic granulomas and fibrosis. Together these data provide, for the first time, a comprehensive overview of the molecular events occurring in the spleen during schistosomiasis and will substantially further our understanding of the local and systemic mechanisms driving the immunopathogenesis of this disease.

Understanding stress-induced immunosuppression: Exploration of cytokine and chemokine gene profiles in chicken peripheral leukocytes

At present, the poultry meat and egg industry has gained a lot of ground, being viewed as a provider of a healthy alternative to red meat and other protein sources. If this trend is to be maintained, solutions must be found to improve resistance of chickens to disease, which often is weakened by stressful conditions. In poultry, stress-induced immunosuppression is manifested by failures in vaccination and increased morbidity and mortality of flocks. Currently, several modern cellular and molecular approaches are being used to explore the status of the immune system during stress and disease. It is likely that these new techniques will lead to the development of new strategies for preventing and controlling immunosuppression in poultry. Using quantitative reverse transcription-PCR assays, a broad spectrum of cytokine, chemokine, and their receptor genes can be quantified in birds and then be used as markers to assess the effects of stress on the immune system. Currently, we are investigating immune and endocrine interactions in the chicken, in particular the cells and molecules that are known to be involved in such interactions in mammals. We have evaluated the effects of corticosterone administration in drinking water on peripheral lymphocyte and heterophil cytokine and chemokine gene profiles. In particular, there seems to be effects on cytokine and chemokine mRNA expression levels in both lymphocytes and heterophils, especially expression of the proinflammatory cytokines interleukin (IL)-1beta, IL-6, and IL-18 and chemokines C-C motif, ligand 1 inflammatory (CCLi1); C-C motif, ligand 2 inflammatory (CCLi2); C-C motif, ligand 5 (CCL5); C-C motif, ligand 16 (CCL16); C-X-C motif ligand 1 inflammatory (CXCLi1); and C-X-C motif ligand 2 inflammatory (CXCLi2), which are initially upregulated and are potentially involved in modulating the adaptive immune response. A chronic treatment with corticosterone downregulates proinflammatory cytokines and chemokines, suggesting that the delayed effects of chronic stress can suppress the immune response. Messenger RNA expression levels of transforming growth factor-beta4 (TGF-beta4) are also upregulated in cortisosterone-treated birds. It appears that the balance between T-helper (Th) 1 and Th2/T regulatory cytokine production is altered in conditions associated with significant changes in plasma corticosterone concentration. Experiments are underway to decipher the cytokine and chemokine responses to vaccination and bacterial challenge on the background of stress-induced immunosuppression.

Human lung innate immune cytokine response to adenovirus type 7

Adenovirus (Ad) type 7 can cause severe infection, including pneumonia, in military recruits and children. The initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. Subsequently, monocytes become evident and, finally, there is a predominantly lymphocytic infiltrate. We have established that Ad7 infection of epithelial cells stimulates release of the neutrophil chemotaxin interleukin (IL)-8, and have extended these studies to a human lung tissue model. Here, we studied cytokine responses to Ad7 in human alveolar macrophages (HAM) and our human lung tissue model. Both ELISA and RNase-protection assay (RPA) data demonstrated that, upon Ad7 infection, IP-10 and MIP-1alpha/beta are released from HAM. IP-10 and MIP-1alpha/beta protein levels were induced 2- and 3-fold, respectively, in HAM 24 h after Ad7 infection. We then investigated induction of specific cytokines in human lung tissue by RPA and ELISA. The results showed that IL-8 and IL-6 were induced 8 h after infection and, by 24 h, levels of IL-8, IL-6, MIP-1alpha/beta and MCP-1 were all increased. IP-10, a monocyte and lymphocyte chemokine, was also induced 30-fold, but only 24 h after infection. Immunohistochemistry staining confirmed that IL-8 was only released from the epithelial cells of lung slices and not from macrophages. IP-10 was secreted from both macrophages and epithelial cells. Moreover, full induction of IP-10 is likely to require participation and cooperation of both epithelial cells and macrophages in intact lung. Understanding the cytokine and chemokine induction during Ad7 infection may lead to novel ways to modulate the response to this pathogen.

Extracellular nucleotides as negative modulators of immunity

Nucleotides are well known for being the universal currency of intracellular energy transactions, but over the past decade it has become clear that they are also ubiquitous extracellular messengers. In the immune system there is increasing awareness that nucleotides serve multiple roles as stimulants of lymphocyte proliferation, ROS generation, cytokine and chemokine secretion: in one word as pro-inflammatory mediators. However, although often neglected, extracellular nucleotides exert an additional more subtle function as negative modulators of immunity, or as immunodepressants. The more we understand the peculiar biochemical composition of the microenvironment generated at inflammatory sites, the more we appreciate how chronic exposure to low extracellular nucleotide levels affect immunity and inflammation. A deeper understanding of this complex network will no doubt help design more effective therapies for cancer and chronic inflammatory diseases.