A new therapeutic approach to treat psoriasis by inhibition of fatty acid oxidation by Etomoxir.

BACKGROUND: The dogma in psoriasis is that due to pathogen-induced inflammatory responses, an autoreactive immune response is induced that leads to tissue destruction. However, this model might be too simplistic. Literature data suggest that the expression of enzymes crucial for fatty acid oxidation is upregulated in the skin of patients with psoriasis compared with healthy individuals. OBJECTIVES: To examine the influence of fatty acid oxidation on psoriasis with regard to expression and activity of the key enzyme in fatty acid oxidation, carnitine palmitoyltransferase-1 (CPT-1) and the effect of the CPT-1 inhibitor, Etomoxir. METHODS: Experiments were performed with homogenates of lesional and healthy skin, fibroblast cultures and a model of human psoriatic skin transplanted on immune-deficient BNX mice. RESULTS: CPT-1 was highly active in lesional skin. Etomoxir was able to block CPT-1 activity in skin, implying that this antagonist may have the potential to suppress psoriasis when administered topically. In the mouse model, Etomoxir had an antipsoriatic effect that was at least as good as that of betamethasone, as evidenced by reduction of epidermal thickness, keratinocyte proliferation and differentiation. CONCLUSIONS: We conclude that fatty acid metabolism and in particular CPT-1 may be an excellent target for treatment of psoriasis.

Physical interaction of human papillomavirus virus-like particles with immune cells.

Human papillomavirus virus-like particles (HPV VLP) and chimeric VLP are immunogens that are able to elicit potent anti-viral/tumor B and T cell responses. To investigate the immunogenicity of VLP, we determined which cells of the immune system are able to bind HPV-16 VLP. VLP were found to bind very well to human and mouse immune cells that expressed markers of antigen-presenting cells (APC) such as MHC class II, CD80 and CD86, including dendritic cells, macrophages and B cells. mAb blocking studies identified Fc gamma RIII (CD16) as one of the molecules to which the VLP can bind both on immune cells and foreskin epithelium. However, transfection of a CD16(-) cell line with CD16 did not confer binding of VLP. Splenocytes from Fc gamma RIII knockout mice showed a 33% decrease in VLP binding overall and specifically to subsets of APC. These combined data support a role for CD16 as an accessory molecule in an HPV VLP-receptor complex, possibly contributing to the immunogenicity of HPV VLP.

Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles.

It has been postulated that upon binding to a cell surface receptor, papilloma virus-like particles (VLPs) gain entry into the cytosol of infected cells and the capsid proteins L1 and L2 can be processed in the MHC class I presentation pathway. Vaccination of mice with human papilloma virus-like particles consisting of capsid proteins L1 and L2 induced a CD8-mediated and perforin dependent protective immune response against a tumor challenge with human papilloma virus transformed tumor cells, which express only minute amounts of L1 protein. Here we show that HPV16 capsid proteins stimulate a MHC class I restricted CTL response with human peripheral blood lymphocytes (PBL) in vitro. The vigorous response was specific for VLP-infected target cells and was MHC class I restricted. Moreover we show the presence of at least one HLA-A*0201 restricted CTL epitope within the HPV-16 capsid proteins by using a VLP-'infected' HLA-A*0201 transfected human cell line as target cells. These results demonstrated that VLPs can induce a HPV16 capsid protein-specific immune response in humans, allowing the monitoring of immune responses induced by vaccines based on chimeric VLPs carrying additional immunogenic peptides or proteins in therapeutical applications in human patients.

Chimeric papillomavirus virus-like particles induce a murine self-antigen-specific protective and therapeutic antitumor immune response.

The use of chimeric virus-like particles represents a new strategy for delivering tumor antigens to the immune system for the initiation of antitumor immune responses. Immunization of DBA/2 mice with the P1A peptide derived from the P815 tumor-associated antigen P1A induced specific T-cell tolerance, resulting in progression of a regressor P815 cell line in all animals. However, immunization with a human papillomavirus type 16 L1 virus-like particle containing the P1A peptide in the absence of adjuvant induced a protective immune response in mice against a lethal tumor challenge with a progressor P815 tumor cell line. Additionally, we demonstrated that these chimeric virus-like particles could be used therapeutically to suppress the growth of established tumors, resulting in a significant survival advantage for chimeric virus-like particle-treated mice compared with untreated control mice. Chimeric virus-like particles can thus be used as a universal delivery vehicle for both tolerizing and antigenic peptides to induce a strong protective and therapeutic antigen-specific antitumor immune response.

Functional analysis of Ran/TC4 as a protein regulating T-cell costimulation.

Antigen (Ag)-triggered activation of T cells requires engagement of both the T-cell Ag receptor and a costimulatory receptor, for which CD28 can function as a prototypical example. CD80 and CD86 represent ligands for this receptor, and although they are present on professional Ag-presenting cells, these molecules are absent from most tumors. Yet some tumors are still able to costimulate a T-cell response, while others cannot. Therefore, a key question concerns the molecular basis for the costimulation of T cells by those tumor cells not expressing the CD28 ligands CD80 and CD86. Upon screening a cDNA library of such a tumor cell line in a transient COS cell transfection assay for costimulatory activity, we identified Ran/TC4 as a protein whose overexpression results in costimulatory activity. Ran/TC4 is a ubiquitously expressed member of the Ras gene superfamily of small guanosine triphosphate-binding proteins and is involved in nuclear transport; Ran/TC4 cDNA-transfected COS cells specifically costimulate CD8 T cells and not CD4 T cells. Transfection of Ran/TC4 into the costimulation-deficient murine RMA lymphoma cell line introduced costimulatory capacity for CD8 T cells and resulted in markedly elevated levels of nuclear Ran/TC4 protein expression. In addition, in vivo priming of mice with Ran/TC4-transfected RMA cells induced protection against wild-type (wt) RMA tumor cells. Ran/TC4-transfected RMA cells and wt RMA tumor cells exhibit comparable in vivo growth rates in mice lacking T and B cells, and Ran/TC4-mediated tumor rejection thus involves B and/or T cells. This possibility is substantiated by the observation that T cells from normal mice challenged with Ran/TC4-transfected RMA cells can mount a cytotoxic T-cell response not only against the Ran/TC4-transfected tumor cells but also against wt RMA tumor cells. Based on these results, we conclude that gene transfer-mediated elevations in Ran/TC4 can confer costimulatory function for CD8 T cells to tumor cells. This finding suggests a novel application of Ran/TC4 as a protein capable of regulating costimulation in tumor cells.

Cellular immunity and immunotherapy against deoxyribonucleic acid virus-induced tumors.

An important role in the immune defense against deoxyribonucleic acid virus induced tumors is mediated by T-cells, as is evident from aetiological, animal model, and clinical data. In this review the most recent observations in this field are described for three prominent members of this family of viruses, namely human papillomavirus associated with human cervical cancer, human adenovirus associated with lung infections in humans and tumors in rodents, and simian virus 40 associated with rodent tumors and human mesothelioma, osteosarcoma and ependymoma.

PBLs of early breast carcinoma patients with a high nuclear grade tumor unlike PBLs of cervical carcinoma patients do not show a decreased TCR zeta expression but are functionally impaired.

Peripheral blood lymphocytes (PBLs) of patients with cervical intraepithelial neoplasia (CIN), cervical carcinoma, or early breast carcinoma were tested for the expression of T cell receptor zeta chain (TCR zeta) and CD16 zeta chain and production of interferon-gamma (IFN gamma) and interleukin (IL) 10. We found that in all patients with CIN and invasive cervical carcinoma, PBLs showed a reduced TCR zeta and CD16 zeta expression and a significant down-regulation in IFN gamma production (a T helper 1 cytokine) after anti-CD3 stimulation. However, the IL 10 secretion (a T helper 2 cytokine) was not diminished after anti-CD3 stimulation. This indicates that only T helper 1 cells are affected by the down-regulation of the TCR zeta chain expression. We also analyzed PBLs of 12 patients with early breast carcinoma. In these patients, we found TCR zeta and CD16 zeta expression down-regulation in 2 of 12 patients. Six of 12 patients had an enhanced TCR zeta expression. The enhanced TCR zeta expression correlated with a reduced IFN gamma expression after anti-CD3 stimulation. These data show that in general PBLs of early breast carcinoma patients, unlike those of cervical carcinoma patients, do not show a decreased TCR zeta expression. However, a functional impairment of T cells was observed in the subgroup of early breast carcinoma patients with a high nuclear grade of their tumor.

CD40 and CD70 co-stimulate a potent in vivo antitumor T cell response.

In several studies, CD80, a potent co-stimulatory molecule, has been reported to be responsible for the induction of CD8+ antitumor T cell responses by CD80-transfected tumor cells. However, expression of CD80 by tumors not always ensures generation of a T cell-mediated antitumor response. Variables such as the inherent immunogenicity of a tumor and its major histocompatibility complex (MHC) expression status affect the efficacy of this approach. Therefore, in this study two other co-stimulatory ligands, CD40 and CD70, have been investigated for their ability to co-stimulate antitumor responses. The efficacy of CD40 and CD70 is compared with that of CD80, with respect to CD4 and CD8 T cell co-stimulatory capacity in vitro and their ability to induce in vivo antitumor responses. Furthermore, CD40 and CD70 are tested for their capacity to induce a long-lived memory response in vivo, as defined both by induction of tumor-specific cytotoxic T lymphocytes (CTLs) and rejection of wild-type tumor cells. It was found that, despite the fact that CD40 predominantly stimulates CD4 T cells, CD40-transfected MHC class II-negative P815 tumor cells become highly immunogenic and induce long-lasting memory tumor-specific CTLs in vivo. Furthermore, CD40 and CD70 emerge as powerful and even superior alternatives to CD80 for improving tumor immunogenicity in vivo. While the mechanisms by which they do so remain to be defined, these findings suggest additional strategies for immunotherapy.

Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model.

Papillomavirus-like particles (VLPs) are a promising prophylactic vaccine candidate to prevent human papillomavirus (HPV) infections and associated epithelial neoplasia. However, they are unlikely to have therapeutic effects because the virion capsid proteins are not detected in the proliferating cells of the infected epithelia or in cervical carcinomas. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, we have generated stable chimeric VLPs consisting of the L1 major capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) nonstructural papillomavirus protein fused to the L2 minor capsid protein. The chimeric VLPs are indistinguishable from the parental VLPs in their morphology and in their ability to agglutinate erythrocytes and elicit high titers of neutralizing antibodies. Protection from tumor challenge was tested in C57BL/6 mice by using the tumor cell line TC-1, which expresses HPV16 E7, but not the virion structural proteins. Injection of HPV16 L1/L2-HPV16 E7 chimeric VLPs, but not HPV16 L1/L2 VLPs, protected the mice from tumor challenge, even in the absence of adjuvant. The chimeric VLPs also induced protection against tumor challenge in major histocompatibility class II-deficient mice, but not in beta2-microglobulin or perforin knockout mice implying that protection was mediated by class I-restricted cytotoxic lymphocytes. These findings raise the possibility that VLPs may generally be efficient vehicles for generating cell-mediated immune responses and that, specifically, chimeric VLPs containing papillomavirus nonstructural proteins may increase the therapeutic potential of VLP-based prophylactic vaccines in humans.

Identification of peptides for immunotherapy of cancer. It is worth the effort.

As the nature of the T cell immune response is defined by T cell receptor recognition of small protein fragments, referred to as peptides, the identification of peptides would lead us to understanding and directing the T-cell-mediated immune response. Immunogenic peptides might be used for vaccination and activation of the immune reaction against cancer- and virus-infected cells. Additionally, the knowledge of immunogenic peptides was expected to lead to blocking of allergic reactions and autoimmune diseases. Based on these assumptions, the search for immunogenic peptides was started in mice and man in the mid-1980s. After a decade of peptide identification and testing in vitro and in vivo, this may be a proper time to evaluate the results from the peptide-related work and determine the possible applications of this knowledge for the next decade. In this review we discuss the identification of peptides, their use in murine models, as well as clinical data from peptide vaccinations or therapies. Potential hazards and limitations of peptide use in immunotherapy and other possible applications for peptides or peptide motifs in immunotherapy are evaluated.

A T cell lymphoma can provide potent co-stimulatory effects to T cells that are not mediated by B7-1, B7-2, CD40, HSA or CD70.

Dominant second signals for T cell activation can be generated through interactions between CD28 and CTLA-4 on T cells with their co-stimulatory ligands B7-1 and B7-2 on APC. Nevertheless, some B7-negative cell lines appear capable of providing second signals to T cells, illustrating that B7-independent co-stimulatory pathways may exist. One such cell line, the peptide-transporter defective T lymphoma RMA-S, was investigated in the present study, to determine the origin of the co-stimulatory effects it provides. RMA-S can support clonal expansion of purified CD4 or CD8 T cells from unprimed mice activated with concanavalin A (ConA) or immobilized anti-CD3. Nevertheless, RMA-S does not express B7-1 or B7-2, nor does it express other known co-stimulatory molecules, i.e. CD40, gp39, CD70 and HSA. Also, co-stimulation provided by RMA-S could not be blocked by antibodies or fusion proteins specific for these co-stimulatory molecules, excluding their participation. However, RMA-S' co-stimulatory activity is dependent on adhesive interactions. RMA-S is incapable of IL-2 production in the presence of ConA or anti-CD3, but T cells co-stimulated by RMA-S produce IL-2 and IFN-gamma upon anti-CD3- or ConA-induced activation. Furthermore, co-stimulation of antigen-specific T cell proliferation of both class I- and class II-restricted T cell clones can be provided by RMA-S, and RMA-S can preclude induction of anergy by 1-ethyl-3-(3-dimethyl amino propyl)carboiimide-fixed APC in a class II-restricted T cell clone. The results suggest that potent co-stimulatory pathways can be induced by cellular interactions between a T lymphoma, RMA-S and T cells, not involving gp39, CD40, CD70, HSA, B7-1 (CD80) or B7-2 (CD86). Characterization of the molecules involved is in progress.

Novel mAbs reveal potent co-stimulatory activity of murine CD27.

Members of the tumor necrosis factor receptor (TNFR) family are emerging as important molecules implicated in the regulation of proliferation, differentiation and survival of T and B lymphocytes. Among these receptors is CD27, the function of which has thus far only been studied in the human system, where it amplifies the T cell proliferative response induced by TCR triggering. We report here the generation of mAbs to murine CD27, by an efficient method involving the use of transfected Armenian hamster fibroblasts. Previous analysis had already indicated that murine CD27 mRNA is uniquely expressed in lymphoid cells. As determined with one of the newly developed antibodies, murine CD27 is expressed on the great majority of both alpha beta and gamma delta T lymphocytes, on a small population of peripheral B cells, and on a very small subset of B220+ cells in the bone marrow. This distribution largely corresponds to that in the human system. However, unlike human CD27, which is primarily expressed in mature, medullary thymocytes, murine CD27 is found on all thymocytes, except a subset of CD4-CD8- precursors. Upon cross-linking, anti-CD27 mAb amplified the proliferative response of purified T lymphocytes to suboptimal stimulation with concanavalin A at least 4-fold. This indicates that such mAbs can mimick ligand binding and demonstrates that CD27 also acts as a potent co-stimulatory molecule in the murine system.

Identification of an H-2 Kb-presented Moloney murine leukemia virus cytotoxic T-lymphocyte epitope that displays enhanced recognition in H-2 Db mutant bm13 mice.

Upon infection with the Moloney murine sarcoma virus-murine leukemia virus (MuLV) complex, H-2b C57BL/6 (B6) mice respond with a class I Db-restricted cytotoxic T-lymphocyte (CTL) response, which protects against virus-induced tumorigenesis. In the B6-derived Db mutant B6.CH-2bm13 (bm13) strain, part of the class I Db antigen-presenting groove is shaped by a class I Kb-encoded sequence. Like B6 mice, bm13 mice reject Moloney virus-induced tumors, but the protective CTL response is Kb restricted. In this study we show enhanced levels of Moloney MuLV-specific CTLp with a restriction for Kb in bm13 mice. Through the use of CTL clones from Moloney virus-immunized bm13 mice, the class I Kb-presented CTL epitope was identified. The epitope is located in the Moloney virus gp70 envelope protein region (Moloney envelope, amino acids 189 to 196 [Mol env (189-196)]), SSWDFITV and has the Kb allele-specific binding motif. The Dbm13 molecule does not present the env(189 to 196) epitope to Kb-restricted bm13 CTL. In B6 mice, Mol env(189-196)-specific CTL could be induced by peptide vaccination. B6 mice thus have CTL precursors specific for this epitope but at considerably lower levels than do bm13 mice. We hypothesize that additional positive selection of Kb-restricted CTL on the Dbm13 molecule in bm13 mice explains this difference in precursor frequencies. We examined related strains of MuLV for the presence of Mol env(189-196) sequence equivalents. Rauscher, Friend, and AKV MuLV-encoded Mol env(189-196) epitope equivalents were properly recognized in cytotoxicity assays, both as synthetic and as endogenously expressed (Rauscher MuLV) peptides. In contrast, the mink cell focus-forming virus MuLV-encoded epitope equivalent, lacking a Kb anchor residue, was not presented for CTL recognition and hence can be excluded as an important CTL epitope for mink cell focus-forming viruses.

Flow cytometric analysis reveals unexpected shared antigens between histologically defined populations of thymic stromal cells.

Utilizing flow cytometry, the expression of antigens recognized by six thymic stromal cell (TSC) reactive mAbs was investigated on fresh TSCs and TSC lines. It was found that some thymic epithelial cells and dendritic cells share antigenic phenotypes, and that most TSC reactive mAbs have a more extensive distribution than would have been predicted from immunohistology. While these findings illustrate the higher sensitivity of flow cytometric analysis, they more importantly emphasize the great complexity of TSC that direct T cell development. In order to identify the molecular parameters that define the various steps involved in T cell differentiation, TSC antigens (non-TCR/MHC/co-receptor) that are functional will have to be identified. This study represents the initial steps in characterizing such antigens.

CD28-B7 interactions are not required for intrathymic clonal deletion.

Activation of antigen specific T cells requires more than stimulation through the TCR-CD3 complex. A second or costimulatory signal is also required, and this second signal can be delivered by interactions between CD28 and B7, ligands expressed on T cells and antigen presenting cells respectively. We have examined the role of the CD28-B7 interaction in superantigen mediated T cell activation and intrathymic negative selection by blocking B7 molecules with a high affinity soluble ligand, CTLA4lg. In vitro T cell activation mediated by both virally encoded endogenous and exogenous bacterial superantigens was significantly blocked by the addition of CTL4Alg to cultures. However, intrathymic clonal deletion in vivo and in fetal thymic organ cultures was not inhibited by blocking B7 molecules. Therefore, although the CD28-B7 costimulation pathway is necessary for T cell activation, it does not appear to play a role in intrathymic clonal deletion.

T cell tolerance and antigen presenting cell function in the thymus.

B7 expression appears much more extensive than previously recognized with anti-human B7 reagents on human leucocyte populations: it is extremely high on splenic and thymic DC, moderate on macrophages and activated B cells, and low on resting B cells. Additionally, B7 is entirely undetectable on any thymic epithelial cells belonging to a panel of transformed cell lines and T cells, but its expression on epithelial cells in situ is still under investigation. This expression pattern is consistent with the hierarchy of costimulatory signal activity among these cell types, with DC being the most effective, and epithelial cells (like other cells of non-hemopoietic origin) not at all. Future studies will investigate to which extent B7 is involved in clonal deletion, i.e., the selection process dependent on self-antigen presentation by DC and B cells.

Mechanisms of induction of primary virus-specific cytotoxic T lymphocyte responses.

We have investigated the ability of various antigen-presenting cell (APC) types to induce primary anti-viral cytotoxic T lymphocyte (CTL) responses by single in vitro stimulation. Of these APC types, only dendritic cells (DC) and RMA-S lymphoma cells could induce primary CTL responses, but by divergent mechanisms. DC were capable of generating primary virus-specific CTL, either by presenting viral peptide or processed infectious virus. In contrast, RMA-S cells could not present endogenous antigen, e.g. after virus infection, but this cell line very efficiently presented exogenous viral peptides to induce primary virus-specific CTL in vitro. Spleen cells, lipopolysaccharide-induced B cell blasts or the non-mutated RMA cells did not have the ability to trigger unprimed T cells by single in vitro stimulation. We have investigated several characteristics important for primary CTL response induction by DC and RMA-S cells (summarized in Fig. 6). Primary CTL response induction by DC or RMA-S cells was blocked by anti-LFA-1 or anti-CD8 monoclonal antibodies (mAb). DC rapidly aggregated with unprimed T cells, which was independent of LFA-1 and CD8 molecules. RMA-S cells did not form conjugates with unprimed T cells. Despite their abundant major histocompatibility complex (MHC) class I cell-surface expression, DC did not bind much exogenously added viral peptide. In contrast, the MHC class I molecules on RMA-S cells bound a large quantity of exogenously administered peptide. Powerful adhesion by DC and high expression of relevant MHC/peptide complexes on RMA-S cells are important features in the initial contact with unprimed T lymphocytes. In a later stage of contact, both DC and RMA-S cells activate LFA-1 (and CD8) molecules at the T cell surface to strengthen and maintain the contact between T cell and APC.

Processing and presentation of intact hen egg-white lysozyme by dendritic cells.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46-61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.

Cytotoxic T lymphocytes against the antigen-processing-defective RMA-S tumor cell line.

RMA-S is an antigen processing-defective cell line, obtained from a Rauscher virus-induced tumor. The cells express only a low level of cell surface major histocompatibility complex (MHC) class I molecules, which are supposed to be devoid of internally derived antigenic peptides. We investigated Rauscher virus expression and Rauscher peptide presentation to virus-specific cytotoxic T lymphocytes (CTL) by this cell line. Viral proteins are expressed properly, both intracellularly and at the cell surface of RMA-S. Rauscher peptides are presented to virus-specific CTL in the groove of both the class I H-2Kb and Db molecules, but at a low level. Culture of RMA-S cells at room temperature increases their susceptibility to CTL. The RMA-S defect thus affects, but not totally abrogates, Rauscher peptide presentation by MHC class I molecules via the endogenous pathway. This indicates that the RMA-S antigen processing deficit is not absolute.