Splenic Antigen-presenting Cells Research Articles

LIVER SINUSOIDAL ENDOTHELIAL CELLS ENDOCYTOSED ALLOGENEIC CELLS TOLERIZE T CELL RECOGNIZING ALLO-ANTIGENS THROUGH INDIRECT RECOGNITION BY FAS-FAS LIGAND PATHWAY.

P953 Although it has been well known that portal injection (PI) of donor cells leads to tolerance to allografts, the mechanism has not been fully understood. The difficulty in determining the precise mechanisms may be due to a difference between tolerizing mechanisms in T cells with direct allospecificity and those with indirect allospecificity. In the present study, we have demonstrated that PI of class II-deficient donor splenocytes led to indefinite acceptance of subsequently transplanted donor-type heart allografts. This finding indicates that PI of donor cells efficiently induces tolerance at least among T cells with indirect allospecificity. PKH-26-labeled B6 splenocytes (30x106 cells/mouse) that were treated with 30-Gy irradiation were injected via the portal vein into Balb/c mice, so that host antigen presenting cells that had taken up the injected splenocytes could be identified as cells that have PKH-26 labeling. At 12 hours after PI, approximately 60% of host liver sinusoidal endothelial cells (LSECs) cells had taken up PKH-26-labeled splenocytes. FCM analyses revealed that naive LSECs were MHC class II+, CD40+, CD80+ and CD86+ phenotypes that are consistent with those of antigen presenting cells (APCs). In contrast, LSECs capturing allogeneic splenocytes lost CD40 expression and inductively expressed Fas-ligand on their surface. To test tolerizing capacity of host LSECs capturing allogeneic splenocytes toward T cells with indirect allospecificity, we examined the effect of antigen presentation by LSECs to naive T cells on the responsiveness of those T cells to subsequent antigen presentation by professional APCs. Balb/c mouse splenocytes first underwent transmigration across the LSECs from Balb/c mice that had been treated with PI of either naïve or irradiated allogeneic B6 splenocytes, enabling direct interaction between T cells and LSECs. The transmigrated T cells, which were predominantly CD4+ T cells, were subsequently stimulated with splenic APCs from Balb/c mice that had been stimulated with intravenous injection of B6 splenocytes. The proliferative response of CD4+ T cells that had transmigrated across the LSECs from mice that had been treated with PI of irradiated splenocytes was significantly reduced compared with that of LSECs from untreated mice. As far as we know, these are the first demonstration that indirect antigen presentation by LSECs contributes to alloreactive T cell tolerance induced by portal injection of donor splenocytes.

Environmental pollutant tributyltin promotes Th2 polarization and exacerbates airway inflammation.

It has been shown that a relatively high dose of tributyltin (TBT), which is recognized as a particularly notable environmental pollutant, exerts immunotoxic effects such as thymic atrophy via induction of T cell apoptosis. However, the effect of low doses of TBT on the immune responses remains unknown. Here we show that environmentally relevant doses of TBT promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4(+) T cells primed with anti-CD3 and splenic antigen-presenting cells (APC). TBT-induced Th2 polarization was indirect, working through APC via suppression of IL-12 production by macrophages/DC and the augmentation of IL-10 production by B cells. Th2 polarization was also induced in mice treated with TBT and immunized with OVA or infected with Nippostrongylus brasiliensis. Furthermore, airway inflammation in mice sensitized and challenged with OVA was exacerbated by the administration of TBT with concomitant augmentation of Th2-type immunity. Our results highlight the fact that an important environmental pollutant TBT may present significant risk for the induction of allergic diseases via promotion of Th2 polarization.

Modification of the Inhibitory Amino Acid for Epitope Peptide Binding onto Major Histocompatibility Complex Class II Molecules Enhances Immunogenicity of the Antigen

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.

Inhibition of hydroxymethylglutaryl-coenzyme a reductase reduces Th1 development and promotes Th2 development.

Several prospective clinical studies have indicated that hydroxymethylglutaryl-coenzyme A reductase inhibitors, statins, prevent cardiovascular events in part through their antiinflammatory properties. Because inflammation is positively and negatively regulated by T helper (Th) 1 cells and Th2 cells, respectively, we examined the effects of statins on the Th polarization in vitro and in vivo. Here we demonstrated that the statins tested, ie, cerivastatin, simvastatin, lovastatin, and atorvastatin, promoted Th2 polarization through both inhibition of Th1 development and augmentation of Th2 development of CD4+ T cells primed in vitro with anti-CD3 antibody and splenic antigen-presenting cells. Cerivastatin exerted most potent effect on modulation of Th1/Th2 development, and the effect was completely abrogated by an addition of mevalonate. Consistent with in vitro experiments, cerivastatin treatment decreased IFN-gamma production of lymph node cells from mice immunized with ovalbumin emulsified in complete Freund's adjuvant, indicating that Th1 development is also suppressed in an in vivo proinflammatory environment. In this murine model, cerivastatin significantly reduced mesangial matrix expansion of glomeruli in the kidney and attenuated proteinuria. The decrease of glomerular sclerosis by cerivastatin treatment was positively related to the suppression of interferon (IFN)-gamma-producing Th1 response in draining lymph node cells. Hence, these findings strongly suggest that statins' inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase regulates Th1/Th2 polarization in vivo and such a mechanism possibly plays a pathophysiological role in immune-related glomerular injury.

CD154–CD40‐independent up‐regulation of B7‐2 on splenic antigen‐presenting cells and efficient T cell priming by staphylococcal enterotoxin A

It has been demonstrated that in vivo T cell priming requires CD154-CD40 interaction, which is suggested to be critical in the induction of co-stimulatory activities on antigen-presenting cells (APC). In the current study, we demonstrate that in vivo administration of a high dose of a superantigen, staphylococcal enterotoxin A (SEA), could up-regulate B7-2 on most splenic APC independently of the CD154-CD40 interaction, followed by efficient expansion of SEA-reactive V(beta)3(+) T cells in CD154- or CD40-deficient mice. However, the CD154-CD40 interaction may be involved in SEA-mediated T cell activation, since a contribution of the CD154-CD40 interaction was observed when a lower dose of SEA was injected. CD154-independent T cell priming by SEA appeared also independent of the TRANCE-RANK pathway, which was shown to be capable of mediating CD154-independent activation of naive T cells during the infection of some viruses. These results indicate that SEA, which provokes rapid and efficient T cell responses without adjuvant, could utilize multiple CD154/TRANCE-independent pathways, to prime T cells.

Total Enantioselective Synthesis and in vivo Biological Evaluation of a Novel Fluorescent BODIPY α‐Galactosylceramide.

ChemInformVolume 34, Issue 14 Natural Products Total Enantioselective Synthesis and in vivo Biological Evaluation of a Novel Fluorescent BODIPY α-Galactosylceramide. Yen Vo-Hoang, Yen Vo-Hoang Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorLaurent Micouin, Laurent Micouin Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorCatherine Ronet, Catherine Ronet Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorGabriel Gachelin, Gabriel Gachelin Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorMartine Bonin, Martine Bonin Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this author Yen Vo-Hoang, Yen Vo-Hoang Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorLaurent Micouin, Laurent Micouin Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorCatherine Ronet, Catherine Ronet Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorGabriel Gachelin, Gabriel Gachelin Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this authorMartine Bonin, Martine Bonin Lab. Chim. Ther., CNRS, Fac. Sci. Pharm. Biol., Univ. R. Descartes, F-75270 Paris, Fr.Search for more papers by this author First published: 20 March 2003 https://doi.org/10.1002/chin.200314228Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume34, Issue14April 8, 2003 RelatedInformation

Total enantioselective synthesis and in vivo biological evaluation of a novel fluorescent BODIPY alpha-galactosylceramide.

Natural killer T (NKT) cells are a distinct subset of mature lymphocytes endowed with features of activated and regulatory T cells. alpha-Galactosylceramides (alpha-GalCers), the synthetic prototype of which is KRN7000, are the only natural reagents recognised by the T-cell receptor of NKT cells. The alpha-GalCer-activated NKT cells promptly release IFN gamma and IL-4 (IFN=interferon; IL=interleukin) and undergo apoptotic death within hours. In mice, activated NKT cells are responsible for antitumour activity and protection against autoimmune diseases. KRN7000 can thus be considered as the root of a family of novel immunoregulatory drugs. To get insights into the in vivo behaviour of alpha-galactosylceramides, an original fluorescent derivative has been prepared by following a convergent synthetic scheme. This strategy allows the introduction of different acyl chains, carbohydrate residues and various labels in the final steps of the synthesis. The fluorescent BODIPY probe derived from a versatile glycolipid precursor is as active as KRN7000 for inducing apoptosis of liver NKT cells. Fluorescence was detected in peritoneal macrophages and splenic antigen-presenting cells, in Kupffer-like cells in the liver, but not in lymphocytes.

Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo

AbstractHigh-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)–based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% ± 6.0%) and spleen (24% ± 3%). Most transduced hepatocytes and nonhepatocytes were nondividing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II–positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo. However, in neonatal recipients, lentiviral transduction was more widespread and included not only hepatocytes and splenic APCs but also cardiomyocytes. The present study suggests potential uses of improved lentiviral vectors for gene therapy of genetic blood disorders resulting from serum protein deficiencies, such as hemophilia, and hepatic disease. However, the use of liver-specific promoters may be warranted to circumvent inadvertent transgene expression in APCs. In addition, these improved lentiviral vectors could potentially be useful for genetic vaccination and treatment of perinatal cardiac disorders.

DCIR Acts as an Inhibitory Receptor Depending on its Immunoreceptor Tyrosine-Based Inhibitory Motif

Major histocompatibility complex class II positive cells, namely dendritic cells, monocytes/macrophages, and B cells, are categorized as antigen-presenting cells. Dendritic cells, so-called professional antigen-presenting cells, use distinct sets of surface receptors before and after maturation: those to capture antigens and those to interact with T cells, respectively. But there remain many surface molecules whose functions are still unknown. In this study, we isolated dendritic cell immunoreceptor from mouse bone-marrow-derived mature dendritic cells. Dendritic cell immunoreceptor is a recently reported C-type lectin receptor characteristic with cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Expression of mouse dendritic cell immunoreceptor mRNA was observed specifically in spleen and lymph node, slightly increased with dendritic cell maturation during in vitro culture of bone marrow cells, and was not detected in cultured natural killer cells. Surface expression of mouse dendritic cell immunoreceptor protein was observed in splenic antigen-presenting cells including B cells, monocytes/macrophages, and dendritic cells, but not in T cells. To reveal the downregulating capacity of dendritic cell immunoreceptor in antigen-presenting cells, the change of B-cell-receptor-mediated signals after coligation with a chimeric Fcgamma receptor IIB containing the cytoplasmic portion of mouse dendritic cell immunoreceptor was examined. As a result, we detected two distinct inhibitory effects of cytoplasmic dendritic cell immunoreceptor minus sign inhibition of B-cell-receptor-mediated Ca2+ mobilization and protein tyrosine phosphorylation minus sign and both of these effects required the tyrosine residue inside the immunoreceptor tyrosine-based inhibitory motif. This report presents immunoreceptor tyrosine-based inhibitory motif-dependent negative regulatory function of dendritic cell immunoreceptors. In conclusion, mouse dendritic cell immunoreceptor expressed on antigen-presenting cells can exert two distinct inhibitory signals depending on its immunoreceptor tyrosine-based inhibitory motif tyrosine residue.

Antigen-presenting cells at the liver tissue in patients with chronic viral liver diseases: CD83-positive mature dendritic cells at the vicinity of focal and confluent necrosis

Although defective functions of peripheral blood or splenic antigen-presenting cells (APCs) are implicated in the pathogenesis of persistent infection in murine and human hepatitis B virus (HBV) and hepatitis C virus (HCV)-carriers, little is known regarding liver-infiltrating APCs in patients with chronic liver diseases. Using immunohistochemical methodology and antigen retrieval technique, we have detected APCs such as HLA DR-positive cells, interdigitating cells (IDCs) and CD83-positive mature and activated dendritic cells (DCs) at the liver specimens from 39 patients with chronic hepatitis (CH) and 10 patients with liver cirrhosis (LC). All 3 types of APCs were detected at the portal areas in both CH and LC, the most abundant being the HLA DR-positive APCs. CD83-positive, mature and activated DCs were detected in patients with CH around the areas of focal and confluent necrosis at the liver parenchyma in close association with T cells. The localization of CD83-positive mature and activated DCs at the liver tissues from patients with CH warrants further study about the role of these DCs in the induction of hepatocellular damage. This may also help to design DC-based therapy for patients with chronic liver diseases.

The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: implications for peptide vaccination.

Targeting of antigens to antigen-presenting cells (APCs) increases CD4(+) T cell activation, and this observation can be exploited in the development of new vaccines. We have chosen an antigen-targeting approach in which we make recombinant antibodies (Abs) with T cell epitopes in their constant region and APC-specific variable regions. Three commonly used model epitopes, amino acids 110-120 of hemagglutinin, 323-339 of ovalbumin, and 46-61 of hen egg lysozyme, were introduced as loops in the C(H)1 domain of human IgG3. For all three epitopes, we show that the recombinant molecules are secreted from transfected cells. The epitopes are presented to specific T cells, and targeting to IgD on B cells in vitro enhances the presentation efficiency by 10(4) to 10(5) compared with the free peptide. After i.v. injection, the epitopes targeted to IgD are presented by splenic APCs to activate specific T cells, whereas little or no activation could be detected without targeting, even after the amount of antigen injected was increased 100-fold or more. Because a wide variety of T cell epitopes, in terms of both length and secondary structure, can be tolerated in loops in constant domains of Abs, the Ab constant region seems to have the intrinsic stability that is needed for this fusion molecule strategy. It might thus be possible to load the Ab with several different epitopes in loops in different domains and thereby make a targeted multisubunit vaccine.

T cell recognition of rat myelin basic protein as a TCR antagonist inhibits reciprocal activation of antigen-presenting cells and engenders resistance to experimental autoimmune encephalomyelitis.

The aim of this study was to assess whether T cell recognition of myelin basic protein (MBP) as a partially antagonistic self antigen regulates the reciprocal activation of professional antigen-presenting cells (APC). This study focused on the rat 3H3 T cell clone that recognized guinea pig (GP) MBP as a full agonist and self rat (R) MBP as a partial agonist. In cultures of 3H3 T cells and splenic APC, the agonist GPMBP elicited several responses by splenic APC, including production of nitric oxide, down-regulation of I-A, induction of B7.1 and B7.2, and prolongation of APC survival. RMBP stimulated a partial increase in production of nitric oxide, partially promoted survival of splenic APC, but did not alter expression of I-A, B7.1, or B7.2 on splenic APC. In the presence ofGPMBP, RMBP antagonized agonist-stimulated induction of B7 molecules, reversed the loss of I-A, and promoted the generation of I-A(+), costimulus-deficient APC. Furthermore, 3H3 T cells cultured with RMBP and irradiated splenocytes reduced the severity of EAE upon adoptive transfer into naive rat recipients subsequently challenged with an encephalitogenic dose of GPMBP/CFA. Overall, this study indicates that T cell receptor antagonism blocks T cell activation, inhibits feedback activation of splenic APC, and promotes T cell-dependent regulatory activities in EAE.

Bone marrow transplant conditioning intensified with liposomal clodronate to eliminate residual host antigen presenting cells fails to ameliorate GVHD and increases PERI-BMT mortality.

Graft versus host disease (GVHD) mediated by allogeneic donor T cells may be initiated and/or exacerbated by residual host antigen presenting cells (APC) which survive the transplant conditioning regimen. We examined whether the depletion of hepatic and splenic APC could reduce the severity of hepatic GVHD after bone marrow transplantation (BMT). Recipient mice were depleted for hepatic and splenic phagocytic APCs by i.v. injection of clodronate- (dichloromethylene diphosphonate) containing liposomes before fully allogeneic or MHC-matched, minor Ag-mismatched BMT. Severity of hepatic GVHD was scored on histological sections 2, 3, 4, or 9 weeks after BMT. No differences in the severity of GVHD were observed between APC-depleted mice and control mice. APC-depleted mice had increased peritransplant mortality due to sepsis. Bacterial clearance assays showed that APC-depleted mice were unable to efficiently clear bacteria, although nondepleted, transplanted mice were able to clear bacteria as quickly as naive control mice. Residual host phagocytic APC do not appear to play a role in the induction of GVHD after BMT. They are, however, essential for prevention of sepsis in the transplant host.

Anti-CD40 Treatment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-Exposed C57Bl/6 Mice Induces Activation of Antigen Presenting Cells Yet Fails to Overcome TCDD-Induced Suppression of Allograft Immunity

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.

UVB irradiation modulates systemic immune responses by affecting cytokine production of antigen-presenting cells.

The immunosuppressive effects of UVB irradiation have been well documented. The production of cytokines by keratinocytes is considered to play a major role in the induction of local as well as systemic immunosuppression. It is thought that partly due to the interaction of locally produced cytokines with antigen-presenting cells (APC) systemic effects, like antigen-specific tolerance, can be induced. In this study we examined the effect of UVB irradiation on cytokine profiles of peripheral APC as well as the functional consequences. Our results indicate that UVB irradiation impairs T(h)1-mediated immune responses in vivo by suppression of the systemic IL-12p70 production. Splenic APC from UVB-exposed mice showed an enhanced production of prostaglandin E(2), IL-1, IL-6 and tumor necrosis factor-alpha after in vitro stimulation. Also, spleen cells from UVB irradiated IL-4(-/-) mice showed increased IL-6 levels. These APC were less efficient in inducing IFN-gamma production by CD4(+) T cells and suppressed IgM production by B cells. We conclude that the altered cytokine profile of peripheral APC can be responsible for the systemic effects of UVB irradiation on the T(h)1/T(h)2 balance as well as on B cell responses.

DNA vaccine delivery by attenuated intracellular bacteria.

Vaccination by intramuscular or intradermal injection of antigen-encoding DNA is a promising new approach leading to strong cellular and humoral immune responses. Since bone-marrow derived antigen presenting cells (APC) seem to induce these immune responses after migration to the spleen, it is desirable to deliver DNA vaccines directly to splenic APC. Recently, attenuated intracellular bacteria have been exploited for the introduction of DNA vaccine vectors into different cell types in vitro as well as in vivo and offer an attractive alternative to the direct inoculation of naked plasmid DNA.

Death by neglect as a deletional mechanism of peripheral tolerance.

In contrast to most organs, the anatomy of the liver may allow naive CD8(+) T cells to make direct contact with liver parenchymal cells. We have previously shown, using a combination of TCR transgenic T cells specific for H-2 K(b) and hepatocytes expressing a transgenic H-2 K(b) molecule, that hepatocytes can induce antigen-specific activation and proliferation of naive CD8(+) T cells independently of CD28 co-stimulation. However, T cell activation by hepatocytes leads to premature T cell death and tolerance, both in vivo and in vitro. In this study, we investigated the mechanisms of T cell death induced by hepatocytes in vitro using primary hepatocytes to activate purified CD8(+) T cells. Neither Fas nor tumor necrosis factor receptor were involved, indicating that hepatocyte- induced death was distinct from activation-induced cell death. Before they started to divide, T cells activated by hepatocytes expressed lower levels of the bcl-x(L) survival gene and 30 times less IL-2 mRNA than CD8(+) cells activated by splenic antigen-presenting cells. Since CD28 co-stimulation increases both IL-2 and bcl-x(L) expression, this suggests that hepatocyte-activated T cells die by neglect because they fail to receive effective co-stimulatory signals. In agreement with this model, premature death promoted by hepatocytes could be prevented by cross-linking CD28. Survival after CD28 cross-linking correlated with increased IL-2 and bcl-x(L) expression, and sustained T cell proliferation, while cytotoxic T lymphocyte activity was prolonged as compared with cells stimulated without CD28 co-stimulation. This study confirms that high- affinity TCR transgenic antigen-specific CD8(+) T cells can be activated to proliferate and differentiate into cytotoxic effector cells. However, prolonged T cell survival and cytotoxicity required CD28 co-stimulation as well. To our knowledge, this is the first report suggesting that tolerance in the context of lack of CD28 co-stimulation can result from Fas-independent peripheral deletion rather than from anergy.

Enhancement of antigen-presenting cell surface molecules involved in cognate interactions by immunostimulatory DNA sequences.

Bacterial genomic DNA, plasmid DNA (pDNA) and synthetic oligodeoxynucleotides (ODN) containing immunostimulatory DNA sequences (ISS) have been proposed to foster a Th1 response via the release of type 1 cytokines from macrophages, dendritic cells, NK cells and B cells. In this study, we show that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (MHC class I and II), co-stimulatory molecules (B7-1, B7-2 and CD40), cytokine receptors (IFN-gamma receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an Fc receptor (Fcgamma receptor) on murine B cells or bone marrow-derived macrophages. The increased expression of these surface molecules is seen in purified cell populations and is largely independent of the effects of type 1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activate naive T cells and bias their differentiation toward a Th1 phenotype in vitro. Thus, the induction of both type 1 cytokines and a distinct profile of cell surface molecules contributes to the potent immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity.

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells.

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide/MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide/MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II/antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP/MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide/MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen/MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide / MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide / MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II / antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP / MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide / MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen / MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.