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Problem statement: Dendritic Cells (DCs) integrate responses of innate and adaptive immune system via transmitting pro-inflammatory and regulatory signals to naive cells within draining lymph nodes. DCs have high capacity for endocytosis, both Clathrin-Dependent (CDE) and Caveolin-Dependent (CvDE). Approach: We have recently shown that ATP-hydrolysis by the multifunctional transporter protein RLIP76 is the primary determinant of the rate of CDE. Activation of CDE is necessary for DCs to internalize and process exogenous antigens. Thus, in present studies we investigated the role of RLIP76 in antigen uptake, maturation and T cell priming capacity of monocyte-derived DCs. Results: Using flow cytometry, we demonstrate variable surface expression of RLIP76 on immature DCs generated from peripheral blood mononuclear cells from healthy individuals. Studies on the effect of anti-RLIP76 antibodies on endocytosis of DC-specific C-type Lectin Receptors (CLRs) (DC-SIGN and DEC-205) and on co-localization of these receptors with MHC-class II were conducted using confocal microscopy. RLIP76-specific antibodies suppressed the maturation of DCs and the expression of typical activation markers and co-stimulatory and adhesion molecules including CD83, HLA-DR, HLA-ABC, CD40, CD80 and CD38. They also inhibited the stimulating potency of DCs in allogeneic Mixed Leukocyte Reaction (allo-MLR). Conclusions: We conclude that RLIP76 is necessary for activation, membrane trafficking and functional maturation of DCs. Further exploration of the role of RLIP76 in DC biology is warranted and may provide promising means in DC-based immunotherapies.

OBJECTIVE Pathology associated with oxidative stress frequently results in insulin resistance. Glutathione (GSH) and GSH-linked metabolism is a primary defense against oxidative stress. Electrophilic lipid alkenals, such as 4-hydroxy-t-2-nonenal (4HNE), generated during oxidative stress are metabolized primarily to glutathione electrophile (GS-E) conjugates. Recent studies show that RLIP76 is the primary GS-E conjugate transporter in cells, and a regulator of oxidative-stress response. Because RLIP76−/− mice are hypoglycemic, we studied the role of RLIP76 in insulin resistance. RESEARCH DESIGN AND METHODS Blood glucose, insulin, lipid measurements, and hyperinsulinemic-euglycemic and hyperglycemic clamp experiments were performed in RLIP76+/+ and RLIP76−/− C57B mice, using Institutional Animal Care and Use Committee–approved protocols. Time-resolved three-dimensional confocal fluorescence microscopy was used to study insulin endocytosis. RESULTS The plasma insulin/glucose ratio was ordered RLIP76−/− < RLIP76+/− < RLIP76+/+; administration of purified RLIP76 in proteoliposomes to RLIP76+/+ animals further increased this ratio. RLIP76 was induced by oxidative or hyperglycemic stress; the concomitant increase in insulin endocytosis was completely abrogated by inhibiting the transport activity of RLIP76. Hydrocortisone could transiently correct hypoglycemia in RLIP76−/− animals, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fructose 1,6-bisphosphatase, in RLIP76−/−. CONCLUSIONS The GS-E conjugate transport activity of RLIP76 mediates insulin resistance by enhancing the rate of clathrin-dependent endocytosis of insulin. Because RLIP76 is induced by oxidative stress, it could play a role in insulin resistance seen in pathological conditions characterized by increased oxidative stress.

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MΦs) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MΦ-derived DCs (ΦDCs). Monocytes were cultured in MΦ-CSF (M-CSF) to produce MΦs, which were then differentiated into DCs following culture with GM-CSF plus IL-4. ΦDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, ΦDCs elicited up to 9-fold more IFN-γ from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not ΦDC IL-12 significantly inhibited T cell IFN-γ induction. ΦDCs produced up to 12-fold more β-chemokines (macrophage-inflammatory protein-1α, -1β, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-γ induction by ΦDCs significantly greater than by MDDCs. Thus DCs differentiating from MΦs induce T cell IFN-γ through β-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-γ induction through chemokines by DCs.

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.

It has been shown that the combined use of two pharmacologic agents can inhibit human immuno­ deficiency virus (HIV) production by peripheral blood mononuclear cells in vitro. One, an anti­ CD25 immunotoxin (IT), kills activated T cellsthat produce virus; the other, the immunosuppressive drug cyclosporine, prevents the quiescent cells, which harbor HIV, from becoming activated. The present study compares the antiviral activities of two agents, SDZ NIM811 and FK506, to that of cyclosporine. In combination with the anti-CD25 IT, these drugs significantly suppressed virus production. In the absence of prior addition of the IT, the ability of the drugs to inhibit virus production was much lower, suggesting that they work effectively in latently infected cells. In the case of SDZ NIM811, the inhibition of virus production was accompanied by a modest inhibition of cell proliferation. In contrast, FK506 exerted strong anti proliferative activity. Cyclosporine was both moderately antiproliferative and a potent antiviral agent.

T cell activation plays a major role in the ability of HIV to remain latent or to establish a productive infection. It has been hypothesized that vaccination-mediated immune stimulation can activate T cells and enhance HIV replication. Our study was designed to determine whether influenza immunization would induce T cell activation and increase HIV burdens in HIV-infected children.Blood samples from 16 HIV-infected children ages 6 months to 14 years were obtained immediately before and 2 and 6 to 8 weeks after the administration of influenza vaccine. The percentage of activated (CD25+) T cells was determined by flow cytometry, and HIV viral load was measured by quantitative cultures of peripheral blood mononuclear cells and plasma HIV RNA.The administration of influenza vaccine was associated with significant increases in HIV viral load in 5 of 16 children evaluated. These increases in HIV burden were transient, and in four of five patients the plasma HIV RNA copy number returned to baseline 6 to 8 weeks after immunization. There was no correlation between the patient's immunologic or clinical category according to the CDC classification and either the initial viral load or the likelihood of having a significant increase after immunization. Four of the five patients who experienced increases in viral load after influenza immunization were not receiving antiretroviral therapy.Our results emphasize the need for additional studies that examine the effect of routine immunizations on T cell activation and HIV replication in HIV-infected children.

RLIP76 is a stress-responsive glutathione-electrophile-conjugates (GS-E) and drugs transporter which is over-expressed in different types of cancers. Cdc2 is a cell-cycle check point control kinase which has been shown to bind to RLIP76 during mitosis, such that endocytosis is inhibited. In present studies, we have purified cdc2 and examined its effect on the transport activity of RLIP76 reconstituted into artificial liposomes. Both doxorubicin (DOX) and dinitro-phenyl S-glutathione (DNP-SG) transport were inhibited by cdc2 in a concentration dependent manner. Liposomal delivery of cdc2 to H358 cells caused apoptosis, resulted in an increased intracellular doxorubicin-accumulation and decreased rate of efflux from the cells. In the present communication, we propose that the accumulation-deficient drug-resistance mediated by RLIP76 can be modulated by inhibition of RLIP76 transport activity by cdc2.

Dendritic cells ignite adaptive immunity by priming naïve T lymphocytes. Human monocyte-derived dendritic cells (MDDCs) infected withToxoplasma gondiiinduce T-lymphocyte gamma interferon production and may thus activateT. gondii-specific immunity. However, we now demonstrate thatT. gondii-infected MDDCs are poor at activating T lymphocytes and are unable to induce specific cytotoxic T lymphocytes. On the other hand, MDDCs acquiring nonviableT. gondiiantigens directly, or indirectly through captured apoptotic or necrotic cell bodies, induce potent T-lymphocyte activation. T lymphocytes exposed to infected MDDCs are significantly impaired in upregulation of CD69 and CD28, are refractory to activation, and die through contact-dependent apoptosis mediated by an as-yet-unidentified mechanism not requiring Fas, tumor necrosis factor-related apoptosis-inducing ligand, leukocyte function antigen 1, intercellular adhesion molecule 1, tumor necrosis factor alpha, interleukin 10, alpha interferon, gamma interferon, prostaglandins, or reactive nitrogen intermediates. Bystander T lymphocytes that were neither infected nor apoptotic were refractory to activation, suggesting global dysfunction. Immunosuppression and T-lymphocyte unresponsiveness and apoptosis are typical of acuteT. gondiiinfection. Our data suggest that infected dendritic cells contribute to these processes. On the other hand, host cells infected withT. gondiiare resistant to multiple inducers of apoptosis. Thus, regulation of host cell and bystander cell apoptosis by viableT. gondiimay be significant components of a strategy to evade immunity and enhance intracellular parasite survival.

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Monocyte-derived dendritic cells (MDDCs) are the most commonly used type of human DC, but are only rarely prepared from mice largely because the yield is small, and there are other easier methods available to produce mouse DCs. Blood is obtained by cardiac puncture. PBMCs are separated by density gradient centrifugation and plated at 8–10 million cells/well in a six-well plate. The mouse peritoneal cavity harbors a small resident population of myeloid cells that are generally regarded as being macrophages. Peritoneal contents are recovered by gentle lavage. Mature mouse DCs will differentiate from fetal liver progenitor cells using a stromal cell feeder layer, GM-CSF, Flt-3L, and SCF to induce differentiation, followed by GM-CSF plus TNFα to induce maturation. DCs isolated from mouse myeloma or GM-CSF and CD40L-transduced colon carcinoma cells will activate tumor-specific T cells without exogenous antigen loading.

The major histocompatibility complex (MHC) class I-related receptor FcRn is involved in regulating serum gammaglobulin (IgG) levels in mice. With the aim of increasing the serum half-life of a recombinant murine Fc gamma 1 fragment, the affinity for binding to FcRn at pH 6.0 has been increased by random mutagenesis of Thr252, Thr254, and Thr256 followed by selection using bacteriophage display. These residues were chosen as they are in proximity to the FcRn-IgG (Fc) interaction site. Two mutants with higher affinity (due to lower off-rates) than the wild-type Fc have been isolated and analyzed in pharmacokinetic studies in mice. The mutant with the highest affinity has a significantly longer serum half-life than the wild type fragment, despite its lower off-rate from FcRn at pH 7.4. The results provide support for the involvement of FcRn in the homeostasis of serum IgGs in mice. The indications that a homologous FcRn regulates IgG levels in humans suggest that this approach has implications for increasing the serum persistence of therapeutic antibodies.

Abstract 3689 Poster Board III-625 Introduction Antigen presentation is, perhaps, the most important function of the immune system for both initiating protective immune response against foreign and/or dangerously altered self structures and maintaining self-tolerance. Dendritic cells (DCs) together with monocytes and macrophages constitute the heterogeneous population of mononuclear phagocytic system and act as non-specific effector cells against microbes and tumors. The unique capacity of DCs to efficiently stimulate naïve T cells, B cells, NK and NKT cells, initiate primary immune response, and orchestrate the intimate interplay between the innate and adaptive arm of the immune system (Banchereau et al, 2000; Whiteside and Odoux, 2004) renders them the most potent professional antigen presenting cells (APCs) (Van Voorhis et al, 1983; Guermonprez et al, 2002). Ral-binding protein 1 (RALBP1), or RLIP76 (Cantor et al, 1995; Jullien-Flores et al, 1995; Park et al, 1995), is a member of the non-ATP-binding cassette (non-ABC) stress-responsive transporters which – in an ATP-dependent fashion – mediates signaling and transport of a wide array of amphiphilic organic substrates such as glutathione-electrophile thioethers (GS-E), genotoxic end-products of lipid peroxidation, e.g., 4-hydroxynonenal (4-HNE), metabolites of arachidonic acid (eicosanoids), natural antineoplastic agents and various xenobiotics. Although mainly involved in mediating efflux of agents normally toxic for the cell, RLIP76 has been identified as primary determinant of the rate of clathrin-coated pit-mediated endocytosis, one of the major ways of antigen uptake and processing by APCs. We, therefore, wanted to investigate the details of this connection, particularly, the significance of changes in cell surface expression of RLIP76 upon DC maturation for the efficiency of antigen uptake and presentation. Methods and Results Using flow cytometry, we found varying cell surface expression of RLIP76 on non-activated (immature) monocyte-derived DCs of healthy individuals, while anti-RLIP76 antibody-treated DCs, even after subsequent delivery of the usual maturation signal, the bacterial lipopolysaccharide (LPS), clearly showed ‘maturation arrest’ as evidenced by low levels of typical activation markers and co-stimulatory molecules, such as CD83, HLA-DR, HLA-ABC, CD80, and CD38. In a functional assay, the antibody-treated DC also failed to fully stimulate allogeneic mixed lymphocyte reaction (allo-MLR) as compared to their untreated counterparts. Conclusions These exciting novel findings predict that expression of RLIP76 on DCs plays an important role in their maturation and that anti-RLIP76 antibodies inhibit this process. Earlier we have also demonstrated that intra-peritoneal administration of RLIP76-loaded proteoliposomes to mice produced increased tissue levels of RLIP76, and, given up to 3 days after irradiation, improved the survival of animals. The above findings led us to hypothesize that augmented presence of RLIP76 in DCs will contribute to improved humoral and cell-mediated immune response through enhanced immune potentiating capacity of DCs. To confirm the implication of these findings in DC-based immunotherapies of human malignancies, more extensive in vitro as well as in vivo experiments, using gene silencing and anti-sense DNA for manipulating expression levels of RLIP76 on DCs, are underway in our laboratory. Disclosures: No relevant conflicts of interest to declare.