Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mphi) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/ME progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.     

Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocyte and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization

We examined the role of the cellular localization of antigen on the immune response after DNA immunization of mice with three forms of ovalbumin (OVA). DNA encoding OVA which was secreted (sOVA) generated 10- to 100-fold higher IgG responses with 50-and 100-fold higher levels of IgG1 than the cytoplasmic (cOVA) or membrane bound (mOVA) forms. An IgG2a predominance was seen only in cOVA and mOVA immunized mice. Although the antibody response was CD4+ T cell dependent, the differences in the antibody response could not be compensated for by provision of excess CD4+ T cell help in TCR transgenic mice. Together with our hapten-carrier studies, this would indicate that membrane or intracellular localization limits the availability of antigen for B cell priming which affects the magnitude and form of the antibody response. Surprisingly, stronger cytotoxic T lymphocyte (CTL) responses were generated for sOVA or mOVA than for cOVA via intramuscular (i.m.) injection. Since a cytoplasmic antigen should have best access to the canonical class I pathway for antigen presentation, our results indicate that priming of CTL responses after i.m. DNA immunization is probably by cross-presentation of antigen by non-transfected professional antigen-presenting cells. In contrast, intradermal immunization with cOVA produced optimal CTL responses but, as with mOVA, suboptimal antibody responses. This, together with our ex vivo RT-PCR analysis showing similar mRNA levels from all three constructs 7 days post-immunization, argues against the differential CTL response for i.m. injection to be due to dose.     

Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for class I antigen presentation

Class I presentation of microinjected native OVA by a temperature-sensitive ubiquitin conjugation mutant, ts85, but not wild-type murine cells, was markedly inhibited following incubation at a nonpermissive temperature. In contrast, the nonpermissive temperature did not affect class I presentation of a minimal OVA peptide expressed in the cytosol. Therefore, these results provide a second example in which a temperature sensitive mutation in the ubiquitin conjugation pathway inhibits MHC class I presentation of native OVA. Surprisingly, incubation at the nonpermissive temperature did not inhibit class I presentation of chemically denatured and alkylated OVA microinjected into the cytosol of mutant cells. Similarly, the presentation of endogenously synthesized OVA (which is expressed from a recombinant vaccinia virus and, presumably, is misfolded in the cytosol) was also not inhibited in both mutant cell lines. Methylation of the lysine groups in denatured OVA, which blocks ubiquitin conjugation, reduced but did not eliminate the presentation of denatured OVA, providing evidence for both ubiquitin-dependent and ubiquitin-independent pathways for class I presentation. In contrast, a proteasome inhibitor blocked class I presentation of all forms of OVA, while a control peptide aldehyde was not inhibitory. These results indicate that modification of the structure of a protein can influence its requirements for ubiquitin conjugation for efficient class I presentation, with the key alteration possibly being the loss of proper conformation. However, regardless of the form of the Ag, the proteasome appears to be required for generating peptides from both endogenously synthesized and microinjected OVA for class I presentation.     

Heavy metal removal by microalgae

The liver is one of the principal sites of iron overload in diseases such as hemochromatosis and beta thalassemia. Hence, much effort has been invested in examining the mechanisms of Fe uptake by hepatocytes. In the present study we have examined the effect of small molecular weight (M(r)) Fe complexes on Fe uptake from iron 59-labeled transferrin (Tf) and 59Fe-labeled citrate by primary cultures of hepatocytes. This was important to assess because Fe-citrate and saturated diferric Tf coexist in the serum of patients with untreated Fe overload. Preincubation of hepatocytes with the low-M(r) Fe complex ferric ammonium citrate (FAC; 25 microg/mL; (Fe) = 4.4 microg/mL) followed by incubation with 59Fe-Tf or 59Fe-citrate ((Fe) = 0.25 to 25 micromol/L) resulted in the marked stimulation of 59Fe uptake. For example, at a physiologically relevant Tf-Fe concentration of 25 micromol/L, there was an 8-fold increase in 59Fe uptake by cells incubated with FAC compared to control cells. In contrast, at Tf-Fe concentrations of 0.25 to 2.5 micromol/L, 59Fe uptake in FAC-treated cells was only 1-fold to 3-fold greater than that in the corresponding controls. These data suggest that the FAC-activated Fe uptake process predominates at physiologically relevant Tf concentrations above the saturation of the Tf receptor (TfR). This is the first study to demonstrate that preincubation of hepatocytes with Iow-M(r)Fe complexes can markedly increase Fe uptake from diferric Tf. In conclusion, these results may help to explain the loading of hepatocytes with Fe that occurs in Fe-overload disease despite marked down-regulation of the TfR.     

Antigen-specific T cell receptor antagonism by antigen-presenting cells treated with the hemolysin of Listeria monocytogenes: a novel type of immune escape

We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II-dependent T cell activation. Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen-presenting cells (APC) with LLO. Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO-treated APC. Incubation of mixtures of two different T cells where only one antigen was presented on LLO-treated APC suggested that T cell inactivation is antigen specific. The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses. This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time. Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.     

GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice

The purpose of this study was to explore whether repeated exposure to aerosolized ovalbumin (OVA) in the context of local expression of GM-CSF can initiate a Th2-driven, eosinophilic inflammation in the airways. On day -1, Balb/c mice were infected intranasally with an adenovirus construct expressing GM-CSF (Ad/GM-CSF). From day 0 to day 9 mice were exposed daily to an OVA aerosol. Mice exposed to OVA alone did not show any evidence of airway inflammation. Mice receiving both Ad/GM-CSF and aerosolized OVA exhibited marked airway inflammation characterized by eosinophilia and goblet cell hyperplasia. Migration of eosinophils into the airway was preceded by a rise in IL-5 and IL-4. Both IL-5 and class II MHC were critically required to generate airway eosinophilia. After resolution, airway eosinophilia was reconstituted after a single OVA exposure. Flow cytometric analysis of dispersed lung cells revealed an increase in macrophages and dendritic cells expressing B7.1 and B7.2, and expansion of activated (CD69-expressing) CD4 and CD8 T cells in mice exposed to OVA and Ad/GM-CSF. Our data indicate that expression of GM-CSF in the airway compartment increases local antigen presentation capacity, and concomitantly facilitates the development of an antigen-specific, eosinophilic inflammatory response to an otherwise innocuous antigen.     

Presentation of a T cell receptor antagonist peptide by immunoglobulins ablates activation of T cells by a synthetic peptide or proteins requiring endocytic processing

T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell-mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (Ig-PLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR-specific T cells may be the operative mechanism for Ig-PLP-LR-mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell-mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.     

Infertility evaluation and treatment according to Jewish law

Nonionic triblock copolymers are relatively nontoxic adjuvants that induce high-titer, long-lasting antibody responses. We have previously shown that these adjuvants also induce cell-mediated immunity including lymphokine production by CD4(+) T cells and cytolytic responses by CD8(+) T cells. These copolymers are thought to modulate hydrophobic adhesive interactions between antigens (Ag) and lymphoid cells. We sought to test the hypothesis that copolymers facilitate uptake of exogenous Ag by antigen-presenting cells (APC) using an in vitro model system. Our data show that nonionic triblock copolymers enhanced presentation of soluble ovalbumin (OVA) to the major histocompatibility complex (MHC) class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells, respectively. Presentation of OVA via the class I pathway was enhanced by copolymers in both phagocytic and nonphagocytic APC. However, copolymers did not enhance binding of peptides to the MHC molecules on APC, presentation of endogenously synthesized Ag, or presentation of exogenous Ag delivered by electroporation. These results provide additional evidence that these nonionic triblock copolymers can serve as powerful adjuvants for augmenting both humoral and cell-mediated immunity to protein Ag.     

Hereditary isolated renal magnesium loss maps to chromosome 11q23

Dendritic cells (DC) expanded in the presence of GM-CSF from the bone marrow of C57BL/6 mice process Gram-negative bacteria expressing the model antigen Crl-OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257-264) processed by DC co-incubated with E. coli expressing Crl-OVA, which contains the Kb-binding OVA(257-264) epitope, occurs by a cytosolic MHC-I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1-/- mice co-incubated with E. coli expressing Crl-OVA did not result in Kb presentation of OVA(257-264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257-264) on Kb when C57BL/6 DC phagocytosed and processed E. coli expressing Crl-OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257-264) processed from bacteria expressing Crl-OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257-264) on neighboring bystander Kb-expressing antigen-presenting cells after BALB/c (H-2d) DC phagocytosed E. coli expressing Crl-OVA could not be detected. Together, these data support a cytosolic MHC-I presentation pathway for OVA(257-264) processed from E. coli expressing Crl-OVA by bone marrow-derived DC.     

Potent differentiation-inducing properties of the antiretroviral agent 9-(2-phosphonylmethoxyethyl) adenine (PMEA) in the rat choriocarcinoma (RCHO) tumor cell model

We have compared the splenic responses following immunization with the T cell-independent (TI)-2 antigen native dextran B512 and with a thymus-dependent (TD) protein-dextran conjugate. Interestingly, primary immunization with native dextran induced germinal center (GC) formation in the spleen to the same extent as the protein-dextran conjugate. The GC were antigen-specific as characterized by the presence of peanut agglutinin (PNA)-positive areas that were also binding FITC-conjugated dextran. Dextran-binding B cells were also detected outside the GC as sites of antibody-producing cells. The secondary splenic response to native dextran was suppressed compared to the primary response, with almost no dextran-specific GC or extra-follicular sites with dextran-specific B cells present in the sections. This suppression could be reverted by using cholera toxin (CT) as an adjuvant for the dextran immunizations. Following native dextran immunization with CT adjuvant a secondary splenic GC response similar to a TD secondary splenic GC response was generated, with almost all the dextran-specific B cells located in the GC. Collectively, this indicates that the difference between TI and TD antigen responses is not due to different abilities in inducing GC development, rather the GC reaction is less productive for a TI antigen than for a TD antigen. CT can both increase secondary GC formation in particular for the TI form of dextran and ameliorate the GC reaction, as reflected by increased anti-dextran antibody levels.     

Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules

By means of the clonotypic variable region, the immunoglobulin (Ig) is a tumor-specific antigen on B cell neoplasms. We report that engagement of the B cell antigen receptor (BcR) promotes presentation of peptides derived from the B cell's intrinsic Ig to major histocompatibility complex (MHC) class II-restricted T cells. Thus, anti-Ig endowed normal, ex vivo B lymphocytes from H-2d, Ig constant heavy chain allotype b (IgCHb) mice with the capacity to stimulate an I-Ad-restricted T cell clone which recognizes the gamma 2ab 435-451 allopeptide. The corresponding self gamma 2aa peptide is cryptic and 6000-fold less antigenic than the gamma 2ab allopeptide. Even so, the syngeneic B cell lymphoma A20 which expresses surface(s) IgG2aa, was also recognized by the T cells after BcR ligation. Thus, anti-Ig triggered the disclosure of a cryptic tumor antigen determinant. We propose that autoantigens, by engaging the BcR of self-reactive B cells, induce presentation of intrinsic Ig peptides to which the T helper cell (Th) repertoire is not tolerant. In this way, B cells with anti-self potential may be activated without Th recognition of nominal autoantigen.     

Epitope cleavage by Leishmania endopeptidase(s) limits the efficiency of the exogenous pathway of major histocompatibility complex class I-associated antigen presentation

The activation of CD8+ T cell responses is commonplace during infection with a number of nonviral pathogens. Consequently, there has been much interest in the pathways of presentation of such exogenous antigens for major histocompatibility complex class I-restricted recognition. We had previously shown that Leishmania promastigotes transfected with the ovalbumin (OVA) gene could efficiently target OVA to the parasitophorous vacuole (PV), with subsequent recognition by class II-restricted T cells. We now report the results of studies aimed at evaluating the PV as a route of entry into the exogenous class I pathway. Bone marrow-derived macrophages can present soluble OVA (albeit at high concentrations) to the OVA(257-264)-specific T cell hybridoma 13.13. In contrast, infection with OVA-transfected Leishmania promastigotes failed to result in the stimulation of this hybridoma. This appeared unrelated to variables such as antigen concentration, parasite survival, and macrophage activation status. These results prompted an analysis of the effects of promastigotes on class I peptide binding using RMA-S cells and OVA(257-264). Our data indicate that the major surface protease of Leishmania, gp63, inhibits this interaction by virtue of its endopeptidase activity against the OVA(257-264) peptide. The data suggest that this activity, if maintained within the PV, would result in loss of the OVA(257-264) epitope. Although we can therefore draw no conclusions from these studies regarding the efficiency of the PV as a site of entry of antigen into the exogenous class I pathway, we have identified a further means by which parasites may manipulate the immune repertoire of their host.     

Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells

Immature dendritic cells (DC) use both macropinocytosis and mannose receptor-mediated endocytosis to internalize soluble Ags efficiently. These Ags are ultimately presented to T cells after DC maturation and migration into the lymph nodes. We have previously described the immortalized myeloid cell line FSDC as displaying the characteristics of early DC precursors that efficiently internalize soluble Ags. To describe the different routes of Ag uptake and to identify the Ag retention compartments in FSDC, we followed the intracellular fate of FITC-coupled OVA, dextran (DX), transferrin, and Lucifer Yellow using flow cytometry, confocal microscopy, and immunoelectron microscopy. OVA and DX gained access into macropinosomes and early endosomes. DX was preferentially sorted into endosomal compartments, while most of the OVA entered macropinosomes via fluid phase uptake. We found a long-lasting retention of DX and OVA of up to 24 h. After 6 h of chase, these two molecules were concentrated in common vesicular compartments. These retention compartments were distinct from endosomes and lysosomes; they were much larger, only mildly acidic, and lacked the small GTP binding protein rab7. However, they were positive for lysosome-associated membrane protein-1, the protease cathepsin D, and MHC class II molecules, thus representing matured macropinosomes. These data suggest that the activity of vacuolar proteases is reduced at the mildly acidic pH of these vesicles, which explains their specific retention of an Ag. The retention compartments might be used by nonlymphoid tissue DC to store peripheral Ags during their migration to the lymph node.     

Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron

The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTB-Fe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTB-Fe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 micromol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 micromol/L) when uptake of iron is TR-mediated, a 10-fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 micromol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax) of Tf-Fe uptake but the Michaelis-Menten constant (Km) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier.     

Interphase cytogenetic (in situ hybridization) analysis of astrocytomas using archival, formalin-fixed, paraffin-embedded tissue and nonfluorescent light microscopy

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.     

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.     

Tingible body macrophages in regulation of germinal center reactions

Tingible body macrophages (TBM), long thought simply as scavengers of apoptotic lymphocytes, are located in the unique microenvironment of germinal centers in close proximity to antigen-retaining follicular dendritic cells (FDC). Observations that TBM endocytose FDC-iccosomal (immune-complex coated bodies) antigen suggested that TBM might present this antigen and help regulate the germinal center reaction. To test for antigen presentation, the ovalbumin (OVA)-specific T(H) hybridoma, 3DO-54.8, which produces IL-2 on receiving effective presentation of OVA, were used as responders to OVA-bearing TBM. Results showed that OVA-bearing TBM failed to induce IL-2 production. Furthermore, addition of TBM to IL-2-inducing positive controls (B cells) not only failed to augment IL-2 production, but rather TBM significantly (55-90%) reduced B-cell induction of IL-2. We found that TBM were rich in prostaglandin by comparison with other nongerminal center lymph node macrophages and that addition of indomethacin to the cultures reversed the inhibitory effect of TBM. Depletion of TBM from enriched preparations, prior to addition to positive control cultures, also abrogated the inhibitory effect on IL-2 production. These data support the concept that TBM, within the unique microenvironment of germinal centers, may be specialized to downregulate the germinal center reaction.     

Retrovirally transduced bone marrow-derived dendritic cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity

It has been extensively documented that murine dendritic cells loaded with tumor-associated Ag (TAA)-derived peptides or protein can prime Ag-specific CD8+ cytotoxic T cells in vivo and can elicit Ag-specific immunity. Optimal presentation of TAA might be achieved by retroviral transduction of DCs allowing long term and stable expression of the TAA-peptides as well as the presentation of multiple epitopes in the context of MHC class I and/or class II molecules. Here we show that retroviral transduction of bone marrow-derived dendritic cells (DCs) with chicken OVA cDNA or the reporter gene green fluorescent protein retained their potent stimulatory capacity and that the transduced DCs could process and present the endogenously expressed OVA protein. The DCs transduced with cDNA encoding native OVA protein presented OVA-derived peptides in the context of MHC class I as well as MHC class II and induced a strong Ag-specific CTL response. DCs expressing a cytosolic form of OVA presented OVA peptides only in the context of MHC class I and failed to induce an OVA-specific CTL response in vivo when they had been cultured in the absence of exogenous protein. Immunization with retrovirally transduced DCs resulted in an Ag-specific immunity and rejection of a tumor cell challenge and a significant survival advantage in tumor-bearing mice. These results obtained in this rapidly lethal tumor model suggest that DCs transduced with TAA may be useful for tumor immunotherapy and underscore the importance of the simultaneous delivery of T cell help in the development of Ag-specific cytotoxic T-cells.     

IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.     

The endocytosis of transferrin by rat intestinal epithelial cells

BACKGROUND/AIMS: The transferrin receptor is a prominent protein on the basal and lateral membranes of intestinal epithelial cells, yet little is known of the function of the receptor in the intestine. The aim of the present study was to determine whether intestinal transferrin receptors were capable of facilitating transferrin internalization. METHODS: Using the rat as an experimental model, the uptake of radiolabeled transferrin by cells isolated from different regions along the crypt-villus axis of the proximal small intestine was studied. RESULTS: An intestinal epithelial cell fraction highly enriched in crypt cells bound most radiolabeled transferrin. Cells in this fraction were able to internalize transferrin and recycle it back to the cell surface. A high affinity, saturable pathway of transferrin uptake by these cells predominated at transferrin concentrations below 0.3 mumol/L, whereas at higher concentrations, most uptake was via a nonsaturable process. Intravenously injected radiolabeled transferrin could be detected within intestinal crypt cells, indicating that these cells are able to internalize transferrin in vivo. CONCLUSIONS: These data suggest that intestinal crypt cells have an active transferrin/transferrin receptor system. Transferrin may play an important role in iron delivery to and/or as a growth factor for the rapidly proliferating intestinal epithelium.