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Biomedical subjects

R E Toes

Publications and source records attributed to R E Toes.

At least 19 recordsLinked to original sources
PubMed2001

Rapid peptide turnover and inefficient presentation of exogenous antigen critically limit the activation of self-reactive CTL by dendritic cells.

This study evaluated to what extent presentation of exogenously acquired self-Ags via MHC class I molecules on DC might contribute to the activation of self-reactive CTL and subsequent development of autoimmune disease. We show here by using the rat insulin promotor lymphocytic choriomeningitis virus glycoprotein model of autoimmune diabetes that the activation of self-reactive CTL by DC after uptake of exogenous Ag is very limited, first by the short half-life of MHC class I-associated peptides on DC in vitro and in vivo, and second by the rather inefficient MHC class I presentation of cell-associated self-Ags by DC. These two mechanisms are probably crucial in establishing high thresholds for the induction of self-reactive CTL that prevent autoimmune sequelae after release of sequestered and previously immunologically ignored tissue Ags.

Amino Acid Sequence↗
PubMed2000

Adoptive T cell immunotherapy of human uveal melanoma targeting gp100.

HLA-A*0201-restricted CTL against human gp100 were isolated from HLA-A*0201/K(b) (A2/K(b))-transgenic mice immunized with recombinant canarypox virus (ALVAC-gp100). These CTL strongly responded to the gp100(154-162) epitope, in the context of both the chimeric A2/K(b) and the wild-type HLA-A*0201- molecule, and efficiently lysed human HLA-A*0201(+), gp100(+) melanoma cells in vitro. The capacity of the CTL to eradicate these tumors in vivo was analyzed in A2/K(b)-transgenic transgenic mice that had received a tumorigenic dose of human uveal melanoma cells in the anterior chamber of the eye. This immune-privileged site offered the unique opportunity to graft xenogeneic tumors into immunocompetent A2/K(b)-transgenic mice, a host in which they otherwise would not grow. Importantly, systemic (i.v.) administration of the A2/K(b)-transgenic gp100(154-162)-specific CTL resulted in rapid elimination of the intraocular uveal melanomas, indicating that anti-tumor CTL are capable of homing to the eye and exerting their tumoricidal effector function. Flow cytometry analysis of ocular cell suspensions with HLA-A*0201-gp100(154-162) tetrameric complexes confirmed the homing of adoptively transferred CTL. Therefore, the immune-privileged state of the eye permitted the outgrowth of xenogeneic uveal melanoma cells, but did not protect these tumors against adoptive immunotherapy with highly potent anti-tumor CTL. These data constitute the first direct indication that immunotherapy of human uveal melanoma may be feasible.

Animals↗
PubMed2000

The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate.

Intracellular protein degradation is a major source of short antigenic peptides that can be presented on the cell surface in the context of major histocompatibility class I molecules for recognition by cytotoxic T lymphocytes. The capacity of the most important cytosolic protease, the 20 S proteasome, to generate peptide fragments with an average length of 7-8 amino acid residues has been thoroughly investigated. It has been shown that the cleavage products are not randomly generated, but originate from the commitment of the catalytically active subunits to complex recognition motifs in the primary amino acid sequence. The role of the even larger 26 S proteasome is less well defined, however. It has been demonstrated that the 26 S proteasome can bind and degrade ubiquitin-tagged proteins and minigene translation products in vivo and in vitro, but the nature of the degradation products remains elusive. In this study, we present the first analysis of cleavage products from in vitro digestion of the unmodified model substrate beta-casein with both the 26 S and 20 S proteasome. The data we obtained show that 26 S and 20 S proteasomes generate overlapping, but at the same time substantially different, sets of fragments by following very similar instructions.

Adenosine Triphosphatases↗
PubMed2000

Immature dendritic cells acquire CD8(+) cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli.

The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.

Animals↗
PubMed2000

Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I-restricted re-presentation of gp96-associated peptides and CTL activation; non-receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Adenovirus E1B Proteins↗
PubMed2000

MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells.

Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-gamma-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192-200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192-200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-gamma-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.

Adenoviruses, Human↗
PubMed2000

The heat shock protein gp96 induces maturation of dendritic cells and down-regulation of its receptor.

Peptides associated with the heat shock protein gp96 induce a specific T cell response against cells from which gp96 is isolated. Recently, we have shown that gp96 binds to a yet unknown receptor present on dendritic cells (DC) and that receptor-mediated uptake is required for cross-presentation of gp96-associated peptides by DC. We now describe that gp96 mediates maturation of DC as determined by up-regulation of MHC class II and CD86 molecules, secretion of the cytokines IL-12 and TNF-alpha and enhanced T cell stimulatory capacity. Heat-denatured gp96 is not able to induce DC maturation and cytokine secretion. Furthermore, we show that mature DC are no longer able to bind gp96 molecules. Hence, the gp96 receptor is down-regulated on mature DC, suggesting that this receptor behaves similar to other receptors involved in antigen uptake like the scavenger receptor CD36, the mannose receptor or the integrins alpha(v)beta(3) and alpha(v)beta(5). Together, our findings provide an additional explanation for the remarkable immunogenicity of gp96 as a cross-priming antigen carrier and direct activator of DC.

Animals↗
PubMed2000

The role of CD40 in peripheral T cell tolerance and immunity.

CD40 and CD40 ligand (CD40L) have been implicated as important molecules for the transformation of nonactivated antigen-presenting cells (APC) into cells that are potent inducers of cytotoxic T lymphocyte (CTL) immunity. The onset of a successful immune response lies within the control of the CD4+ T helper cells which, after specific antigen recognition, can up-regulate CD40L and subsequently activate APC through CD40 signaling. Triggering of CD40 with antibodies in vivo can replace the need for CD40L-expressing CD4+ T helper cells for cross-priming of CTL. Blocking of CD40-CD40L interactions can also have profound effects on the generation of T cell immunity. Interestingly, differential involvement of CD40/CD40L in immune responses can be observed between various immunological sites in the body. In most sites of the periphery interruption of CD40-CD40L interactions can lead to the induction of T cell tolerance whereas in mucosal tissues this interruption can lead to abrogation of T cell tolerance. Furthermore, in vivo CD40 activation can convert specific T cell tolerance following peptide vaccination into efficient T cell priming. Thus intervention of CD40-CD40L interactions can result in enhancement or down-modulation of T cell reactivity and therefore modulation of these interactions may form the foundation of new treatment modalities directed against malignancies, allergies, organ rejections and autoimmunity.

Animals↗
PubMed2000

Design and evaluation of antigen-specific vaccination strategies against cancer.

After studies in preclinical mouse models, the efficacy and safety of tumor-specific vaccination strategies is currently being evaluated in cancer patients. The first wave of clinical trials has shown that in general such vaccination strategies are safe. However examples of clinical responses, especially in conjunction with vaccine-induced immune responses, are still scarce. The fact that most trials have so far been performed with end-stage cancer patients can largely account for this deficit. Greater efficacy of anticancer vaccines is expected in patients with less-progressed disease. In addition, the detection of both natural and vaccine-induced T cell immunity needs further improvement.

Antigens↗
PubMed2000

The heat shock protein gp96: a receptor-targeted cross-priming carrier and activator of dendritic cells.

Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by upregulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-alpha and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.

Animals↗
PubMed1999

Successful immunotherapy of an intraocular tumor in mice.

Immune privilege in the eye is considered essential in the protection against local sight-threatening inflammatory responses. However, the deviant immune responses in the eye may also provide an ideal opportunity to uncontrolled growth of viruses or tumors by inhibiting intraocular immunological attack. To establish to what extent immune privilege interferes with T cell-mediated antitumor immunotherapy, we established a new ocular tumor model in the mouse and tested whether well-defined tumor-specific CTLs can eradicate an immunogenic intraocularly growing tumor. Tumor cells, transformed by human adenovirus type 5 early region 1 (Ad5E1), injected s.c. in a dose of 10(7) cells, did not induce s.c. tumor growth in C57BL/6 mice. However, an injection of 0.3 x 10(6) of these cells into the anterior chamber of the eye led to intraocular tumor growth in 95% of mice (n = 20). Tumor growth in the eye did not induce systemic tumor-specific tolerance, because 70% of the mice were able to eradicate the tumor spontaneously after 5 weeks. Mice vaccinated s.c. with irradiated tumor cells were protected against intraocular tumor challenge, indicating that preactivated memory T cells are able to protect against intraocular tumor growth. Moreover, an i.v. injection of an Ad5E1-specific CTL clone was able to eradicate established intraocular Ad5E1-transformed tumors, whereas the anatomy of the eye remained intact. These results demonstrate that tumor-specific, CTL-mediated immunity can be used successfully for the prevention and eradication of tumors growing in the immune-privileged anterior chamber of the eye, without detectable destruction of the eye.

Adenoviruses, Human↗
PubMed1999

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.

Adenovirus E1A Proteins↗
PubMed1999

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset.

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.

Adenoviruses, Human↗
PubMed1998

Cross-priming of CTL responses in vivo does not require antigenic peptides in the endoplasmic reticulum of immunizing cells.

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.

Adenovirus E1A Proteins↗