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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

Rejection of intraocular tumors by CD4(+) T cells without induction of phthisis.

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4(+) T cells. We now show that intraocular tumors can be eradicated by CD4(+) Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8(+) T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-alpha, Fas ligand, MHC class I, and CD8(+) T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4(+) Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4(+) T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.

Adenovirus E1 Proteins↗
PubMed2001

Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+) regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses.

Therapeutic efficacy of a tumor cell-based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte-associated antigen (CTLA)-4 pathway or the CD25(+) regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25(+) Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2(180-188)-specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8(+) T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25(+) Treg cells indicates that CD25(+) Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity.

Abatacept↗
PubMed2001

Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells.

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.

Animals↗
PubMed2001

Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance.

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.

Adenovirus E1A Proteins↗
PubMed2001

Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products.

Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon gamma--inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex--like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously. We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell--specificity as indicated by the observation that LMP-7(-/-) mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.

Amino Acid Sequence↗
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↗
PubMed2001

Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells.

Inhibitory natural killer receptors (NKRs) such as killer cell immunoglobulin-like receptors (KIRs) in humans and Ly49 molecules in mice are expressed on NK cells and recognize multiple major histocompatibility (MHC) class I proteins. In humans and mice, a subset of CD8+ T cells also expresses NKRs and harbors a memory phenotype. Using mice that are transgenic for KIR2DL3 and its cognate HLA-Cw3 ligand, we show that engagement of inhibitory NKRs selectively drives the in vivo accumulation of a subset of memory-phenotype CD8+ T cells that express the beta chain of the interleukin 2 receptor. In vitro, recognition of MHC class I molecules by inhibitory NKRs on T cells down-regulated activation-induced cell death. These results unveil an MHC class I-dependent pathway that promotes the survival of a subset of memory-phenotype CD8+ T cells and also reveal an unexpected biological function for inhibitory NKRs on T cells.

Animals↗
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↗