Transplacental Antibody Transfer of Respiratory Syncytial Virus Specific IgG in Non-Human Primate Mother-Infant Pairs.

One approach to protect new-borns against respiratory syncytial virus (RSV) is to vaccinate pregnant women in the last trimester of pregnancy. The boosting of circulating antibodies which can be transferred to the foetus would offer immune protection against the virus and ultimately the disease. Since non-human primates (NHPs) have similar reproductive anatomy, physiology, and antibody architecture and kinetics to humans, we utilized this preclinical species to evaluate maternal immunization (MI) using an RSV F subunit vaccine. Three species of NHPs known for their ability to be infected with human RSV in experimental challenge studies were tested for RSV-specific antibodies. African green monkeys had the highest overall antibody levels of the old-world monkeys evaluated and they gave birth to offspring with anti-RSV titers that were proportional to their mother. These higher overall antibody levels are associated with greater durability found in their offspring. Immunization of RSV seropositive AGMs during late pregnancy boosts RSV titers, which consequentially results in significantly higher titers in the vaccinated new-borns compared to the new-borns of unvaccinated mothers. These findings, accomplished in small treatment group sizes, demonstrate a model that provides an efficient, resource sparing and translatable preclinical in vivo system for evaluating vaccine candidates for maternal immunization.

Decreased complexity of glucose dynamics in diabetes in rhesus monkeys.

Until recently, preclinical and clinical work on diabetes has focused on the understanding of blood glucose elevation and its detrimental metabolic sequelae. The advent of continuous glucose monitoring (CGM) technology now allows real time monitoring of blood glucose levels as a time series, and thus the exploration of glucose dynamics at short time scales. Previous work has shown decreases in the complexity of glucose dynamics, as measured by multiscale entropy (MSE) analysis, in diabetes in humans, mice, and rats. Analyses for non-human primates (NHP) have not been reported, nor is it known if anti-diabetes compounds affect complexity of glucose dynamics. We instrumented four healthy and six diabetic rhesus monkeys with CGM probes in the carotid artery and collected glucose values at a frequency of one data point per second for the duration of the sensors' life span. Sensors lasted between 45 and 78 days. Five of the diabetic rhesus monkeys were also administered the anti-diabetic drug liraglutide daily beginning at day 39 of the CGM monitoring period. Glucose levels fluctuated during the day in both healthy and diabetic rhesus monkeys, peaking between 12 noon - 6 pm. MSE analysis showed reduced complexity of glucose dynamics in diabetic monkeys compared to healthy animals. Although liraglutide decreased glucose levels, it did not restore complexity in diabetic monkeys consistently. Complexity varied by time of day, more strongly for healthy animals than for diabetic animals. And by dividing the monitoring period into 3-day or 1-week subperiods, we were able to estimate within-animal variability of MSE curves. Our data reveal that decreased complexity of glucose dynamics is a conserved feature of diabetes from rodents to NHPs to man.

A novel method for strict intranasal delivery of non-replicating RSV vaccines in cotton rats and non-human primates.

Respiratory syncytial virus (RSV) is the most common viral cause of bronchiolitis and pneumonia in children twelve months of age or younger and a significant cause of lower respiratory disease in older adults. As various clinical and preclinical candidates advance, cotton rats (Sigmodon hispidus) and non-human primates (NHP) continue to play a valuable role in RSV vaccine development, since both animals are semi-permissive to human RSV (HRSV). However, appropriate utilization of the models is critical to avoid mis-interpretation of the preclinical findings. Using a multimodality imaging approach; a fluorescence based optical imaging technique for the cotton rat and a nuclear medicine based positron emission tomography (PET) imaging technique for monkeys, we demonstrate that many common practices for intranasal immunization in both species result in inoculum delivery to the lower respiratory tract, which can result in poor translation of outcomes from the preclinical to the clinical setting. Using these technologies we define a method to limit the distribution of intranasally administered vaccines solely to the upper airway of each species, which includes volume restrictions in combination with injectable anesthesia. We show using our newly defined methods for strict intranasal immunization that these methods impact the immune responses and efficacy observed when compared to vaccination methods resulting in distribution to both the upper and lower respiratory tracts. These data emphasize the importance of well-characterized immunization methods in the preclinical assessment of intranasally delivered vaccine candidates.

Direct stimulation of T lymphocytes by immunosomes: virus-like particles decorated with T cell receptor/CD3 ligands plus costimulatory molecules.

Many infectious viruses coevolved with the vertebrate immune system. During the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, called lipid rafts, frequently function as a natural meeting point for viral proteins. The role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and T cell signaling is widely recognized. In our studies, we determined whether lipid rafts, virus budding, and molecular interactions during T cell activation could be brought into a novel context to create artificial antigen-presenting particles. We show here that cell-free virus-like particles (VLP) expressing a surrogate TCR/CD3 ligand (OKT3scFv) and the costimulator CD80 polyclonally activate human T cells independently of accessory cells. VLP expressing the glycoprotein epitope 33-41 of the lymphocytic choriomeningitis virus in the context of H-2D(b) activate and expand naïve, antigen-specific CD8(+) T lymphocytes and differentiate them into cytotoxic effector cells. Efficient targeting of T cell ligands to lipid rafts and ultimately to VLP is achieved by C-terminal introduction of glycosyl phosphatidyl inositol acceptor sequences, replacing transmembrane and intracellular domains. In this work, basic functions of immunostimulatory molecules meet virus biology and translate into a reductionist antigen-specific T lymphocyte-stimulating vehicle, which we refer to as immunosomes. A large variety of agonistic and antagonistic accessory molecules on genuine antigen-presenting cells may complicate the predictable manipulation of T cells as well as the analysis of selected receptor combinations, making immunosomes potentially useful reagents for such purposes in the future.

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression.

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.

Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40.

Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cytokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation.

CD63 as an activation-linked T cell costimulatory element.

Dendritic cells (DC) are unique in their capacity to either stimulate or regulate T cells, and receptor/ligand pairs on DC and T cells are critically involved in this process. In this study we present such a molecule, which was discovered by us when analyzing the functional effects of an anti-DC mAb. This mAb, 11C9, reacted strongly with DC, but only minimally with lymphocytes. In MLR it constantly reduced DC-induced T cell activation. Therefore, we assumed that mAb 11C9 primarily exerts its functions by binding to a DC-structure. This does not seem to be the case, however. Preincubation of DC with mAb 11C9 before adding T cells had no inhibitory effect on T cell responses. Retroviral expression cloning identified the 11C9 Ag as CD63. This lysosomal-associated membrane protein (LAMP-3), is only minimally expressed on resting T cells but can, as we show, quickly shift to the surface upon stimulation. Cross-linkage of that structure together with TCR-triggering induces strong T cell activation. CD63 on T cells thus represents an alternative target for mAb 11C9 with its binding to activated T cells rather than DC being responsible for the observed functional effects. This efficient CD63-mediated costimulation of T cells is characterized by pronounced induction of proliferation, strong IL-2 production and compared with CD28 enhanced T cell responsiveness to restimulation. Particularly in this latter quality CD63 clearly surpasses several other CD28-independent costimulatory pathways previously described. CD63 thus represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion.

Initial human myeloid/dendritic cell progenitors identified by absence of myeloperoxidase protein expression.

Myeloperoxidase (MPO) represents an early-appearing and highly reliable intracellular myeloid lineage marker molecule. MPO protein can be detected in a subset of human hematopoietic bone marrow progenitor cells and in granulomonopoietic (GM) cells. However, other myeloid-related cell types such as epidermal Langerhans-type dendritic cells (LC) lack MPO. Therefore, human myeloid progenitors might be subdivided based on MPO protein expression into functional subsets. Here we identified two consecutive myelopoietic cell stages, i.e., early myeloid progenitors that lack MPO, as well as their immediate MPO+ progeny. MPO- myeloid progenitors possess previously described granulomonocyte (GM) progenitor-associated cell-surface characteristics (CD34+CD45RA+CD13+lin-). They are specifically recruited and can be expanded in cultures of CD34+ cord blood cells in response to early-acting hematopoietic cytokines. Furthermore, cell fractions enriched in MPO- myeloid progenitors efficiently developed along Langerhans-type dendritic cell (LC) and granulomonocytic (GM) lineages, whereas progeny enriched in MPO+ cells showed diminished LC potential. In line with this, peripheral blood progenitors, known to possess LC differentiation potential, lacked MPO expression. We conclude that differential expression of MPO therefore further characterizes cells with myeloid or LC potential.

Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains.

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-gamma production.

Fish oil interaction with warfarin.

OBJECTIVE: To report a case of elevated international normalized ratio (INR) in a patient taking fish oil and warfarin. CASE SUMMARY: A 67-year-old white woman had been taking warfarin for 1(1/2) years due to recurrent transient ischemic attacks. Her medical history included hypothyroidism, hyperlipidemia, osteopenia, hypertension, and coronary artery disease. She also experienced an inferior myocardial infarction in 1995 requiring angioplasty, surgical repair of her femoral artery in 1995, and hernia repair in 1996. This patient has her INR checked in the anticoagulation clinic and is followed monthly by the clinical pharmacist. Prior to the interaction, her INR was therapeutic for 5 months while she was taking warfarin 1.5 mg/d. The patient admitted to doubling her fish oil dose from 1000 to 2000 mg/d. Without dietary, lifestyle, or medication changes, the INR increased from 2.8 to 4.3 within 1 month. The INR decreased to 1.6 one week after subsequent fish oil reduction, necessitating a return to the original warfarin dosing regimen. DISCUSSION: Fish oil supplementation could have provided additional anticoagulation with warfarin therapy. Fish oil, an omega-3 polyunsaturated fatty acid, consists of eicosapentaenoic acid and docosahexaenoic acid. This fatty acid may affect platelet aggregation and/or vitamin K-dependent coagulation factors. Omega-3 fatty acids may lower thromboxane A(2) supplies within the platelet as well as decrease factor VII levels. Although controversial, this case report illustrates that fish oil can provide additive anticoagulant effects when given with warfarin. CONCLUSIONS: This case reveals a significant rise in INR after the dose of concomitant fish oil was doubled. Patients undergoing anticoagulation therapy with warfarin should be educated about and monitored for possible drug-herb interactions. Pharmacists can play a crucial role in identifying possible drug interactions by asking patients taking warfarin about herbal and other alternative medicine product use.

MHC class II antigen signaling induces homotypic and heterotypic cluster formation of human mature monocyte derived dendritic cells in the absence of cell death.

Cellular aggregation in response to surface antigen signaling is a regulated process important for cell-cell interaction and cell migration. We studied dendritic cell (DC) aggregation in response to major histocompatibility complex (MHC) class II antigen ligation using human monocyte derived mature DCs. Crosslinking of MHC class II antigens by monoclonal antibodies in DCs matured by different stimuli, but not in immature DCs, induced the formation of large and long-lived homotypic cell clusters within 1 hour. These aggregates were completely resistant to mechanical disruption and displayed no signs of increased cell death. Heterotypic conjugate formation of mature DCs with lymphocytes and monocytes following addition of MHC class II antibodies occurred as well. DC aggregation required antigen dimerization, the presence of serum and energy, an intact cytoskeleton, and could not be blocked by EDTA, mannan, and monoclonal antibodies specific for integrins. These data underscore the positive role of MHC class II signaling in mature DCs suggesting the possibility of reverse DC activation in the course of antigen specific interaction with lymphocytes.

SLAM-associated protein deficiency causes imbalanced early signal transduction and blocks downstream activation in T cells from X-linked lymphoproliferative disease patients.

Deficiency of SAP (SLAM (signaling lymphocyte activation molecule)-associated protein) protein is associated with a severe immunodeficiency, the X-linked lymphoproliferative disease (XLP) characterized by an inappropriate immune reaction against Epstein-Barr virus infection often resulting in a fatal clinical course. Several studies demonstrated altered NK and T cell function in XLP patients; however, the mechanisms underlying XLP disease are still largely unknown. Here, we show that non-transformed T cell lines obtained from XLP patients were defective in several activation events such as IL-2 production, CD25 expression, and homotypic cell aggregation when cells were stimulated via T cell antigen receptor (TCR).CD3 but not when early TCR-dependent events were bypassed by stimulation with phorbol 12-myristate 13-acetate/ionomycin. Analysis of proximal T cell signaling revealed imbalanced TCR.CD3-induced signaling in SAP-deficient T cells. Although phospholipase C gamma 1 phosphorylation and calcium response were both enhanced in T cells from XLP patients, phosphorylation of VAV and downstream signal transduction events such as mitogen-activated protein kinase phosphorylation and IL-2 production were diminished. Importantly, reconstitution of SAP expression by retroviral-mediated gene transfer completely restored abnormal signaling events in T cell lines derived from XLP patients. In conclusion, SAP mutation or deletion in XLP patients causes profound defects in T cell activation, resulting in immune deficiency. Moreover, these data provide evidence that SAP functions as an essential integrator in early TCR signal transduction.

Differential methylation pattern of the X-linked lymphoproliferative (XLP) disease gene SH2D1A correlates with the cell lineage-specific transcription.

SH2D1A, the X-linked lymphoproliferative disease (XLP) gene, encodes a cytoplasmic protein that plays an essential role in controlling Epstein-Barr virus infection. It is expressed in T and NK cells, but not in B cells or in granulocytes. The promoter, the regulatory regions, as well as the mechanisms controlling its tissue-specific expression, are still unknown. We tested the hypothesis that DNA methylation might contribute to tissue-specific SH2D1A gene expression and analyzed the methylation status of 2,300 bp upstream of the ATG starting codon, the coding region and part of intron 1. By bisulfite sequencing and methylation-sensitive restriction enzyme digestion, we show that a differential methylation pattern of CpG-rich regions in the 5' region and the adjacent exon 1 of the SH2D1A gene indeed correlates with the tissue-specific gene transcription.

B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy.

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.

Retrovirus-mediated IL-7 expression in leukemic dendritic cells generated from primary acute myelogenous leukemias enhances their functional properties.

Myeloid lineage-derived dendritic cells (DCs) are considered the professional antigen-presenting cell type responsible for eliciting T-cell-mediated immune responses. Acute myelogenous leukemia (AML) is a disease in which tumor antigens are expressed by the malignant clone that also has the potential to differentiate into DC-like cells (leukemic DCs) with antigen-presenting capacity. This study investigated whether the constitutive expression of the cytokine interleukin-7 (IL-7) in primary AML cells during their differentiation toward leukemic DCs results in superior antigen-presenting cells. A bicistronic retroviral vector encoding the IL-7 cytokine and the surface immunoselectable low-affinity nerve growth factor receptor (LNGFr) gene was constructed and used for transduction experiments. A serum-free system was used to transduce and differentiate leukemic cells toward leukemic DCs. The study included 8 patients with AML. The transduction efficiency with the cytokine vector varied among patients, ranging from 5% to 30% as judged by LNGFr expression. The leukemic origin of the transduced cells was confirmed in a patient with a chromosomal translocation t(9:11) by fluorescence in situ hybridization analysis. Cytokine modified-cells consistently secreted IL-7 (mean, 415 pg +/- 190/10(6) cells/48 hours; n = 5). We demonstrate that IL-7-transduced cells are included in the differentiated leukemic DC subset, and, as shown in a particular case, that about half of the mature CD80(+) and CD83(+) populations coexpress the LNGFr transgene. In addition, IL-7-modified leukemic cells induce stronger allo-T-cell stimulation and higher amounts of IL-2 production in T cells compared with control groups. Finally, cytokine-transduced leukemic DCs can effectively prime and generate cytotoxic T lymphocytes against autologous leukemic blasts.

Monoclonal Antibodies to Human Cell Surface Antigens.

This appendix lists the cluster of differentiation (CD) designations for the nearly 300 different leukocyte surface molecules that have been assigned and approved by the nomenclature committee of the International Union of Immunological societies (IUIS).

Cross-priming of cytotoxic T cells promoted by apoptosis-inducing tumor cell reactive antibodies?

Humanizing xenogenic monoclonal antibodies (MAbs) by genetic engineering has greatly improved their therapeutic utility and efficacy. The chimeric CD20 MAb C2B8 (Rituximab) is a prominent representative of this new generation of therapeutic MAbs and has been proposed as a treatment of choice for recurrent follicular non-Hodgkin's lymphomas. Treatment of CD20+ B cells with MAb C2B8 triggers several cell-damaging actions including complement-mediated lysis (CDL), antibody-dependent cellular cytotoxicity (ADCC), and MAb-induced induction of apoptosis. We provide an overview of the most prominent mechanisms underlying the efficacy of antibody treatment. We introduce our concept of cross-priming of cytotoxic T-cell responses promoted by apoptosis incucing antibodies. Treatment of tumor cells with antibodies that are capable of inducing a proapoptotic signal via their cell surface target structure may not only contribute to their direct killing but also may induce cellular responses against the tumor, which may have a long-lasting protective effect. We report, using the example of C2B8 anti-CD20 treatment of lymphoma cells, that MAb C2B8-induced apoptosis of lymphoma cells not only kills these cells but also promotes uptake and cross-presentation of lymphoma cell-derived peptides by antigen-presenting dendritic cells (DC), induces maturation of DC, and allows the generation of specific CTL.

Identification of human CD93 as the phagocytic C1q receptor (C1qRp) by expression cloning.

CD93 is a approximately 120 kDa O-sialoglycoprotein that within the hematopoietic system is selectively expressed on cells of the myeloid lineage. So far, its primary structure and function were unknown. We used retroviral-expression cloning to isolate the CD93 cDNA. Sequence analysis revealed that CD93 is identical to a protein on human phagocytes termed C1q receptor (C1qRp). C1qRp was shown previously to mediate enhancement of phagocytosis in monocytes and was suggested to be a receptor of C1q and two other structurally related molecules. When studying CD93 transductants and control cells, we found that cells expressing CD93 have enhanced capacity to bind C1q. Furthermore, we show that immature dendritic cells (DC) express CD93/C1qRp, and mature DC, known to have reduced capacity for antigen uptake and to have lost the ability to phagocytose, show weak-to-negative CD93/C1qRp expression.