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.     

Costimulation, tolerance and ignorance of cytolytic T lymphocytes in immune responses to tumor antigens

Despite the fact that many tumors express MHC class I molecules presenting "foreign" peptide antigens, a vigorous tumor-destructing immune response is seldom detected. A possible explanation is that tumors cannot provide adequate costimulatory signals as provided by professional antigen presenting cells. CD28, upon interacting with B7, triggers costimulatory signals critical for the T-cell response. Transfection of tumor cells with B7 augments the immunogenicity of the tumor so that an anti-tumor immune response can be amplified. When B7-CD28 costimulation is provided CTL specific for otherwise silent epitopes can be activated. Therefore, unresponsiveness of T cells to many tumor antigens should be considered as ignorance rather than tolerance. Immunological ignorance may thus contribute to the failure of the immune system to respond against the tumor antigens.     

Schiff base forming drugs: mechanisms of immune potentiation and therapeutic potential

CD4 T-lymphocytes, which orchestrate immune responses, receive a cognitive signal when clonally distributed receptors are occupied by MHC class II bound peptides on antigen-presenting cells. The latter provide costimulatory or accessory signals through macromolecules such as B7.1 and B7.2 which interact with coreceptors on T-cells to regulate outcomes in terms of T-cell activation or specific non-responsiveness. Complementary studies at the chemical level have implicated Schiff base formation between specialised carbonyls and amines, constitutively expressed on antigen-presenting cell and T-cell surfaces, as an essential element in specific T-cell activation. The small xenobiotic Schiff base forming molecule tucaresol, which substitutes for the physiological donor of carbonyl groups to provide a costimulatory signal to CD4 T-helper lymphocytes (Th-cells), has been developed for testing as an immunopotentiatory drug. Tucaresol, which is orally bioavailable and systemically active, enhances CD4 Th-cell and CD8 cytotoxic T-cell responses in vivo and selectively favours a Th1-type profile of cytokine production. In murine models of virus infection and syngeneic tumour growth it has substantial therapeutic activity. Schiff base formation by tucaresol on T-cell surface amines provides a costimulatory signal to the T-cell through a mechanism that activates clofilium-sensitive K+ and Na+ transport. The signalling pathway utilised by tucaresol converges with T-cell receptor signalling at the level of MAP kinase, promoting the tyrosyl phosphorylation of ERK2 by MEK (mitogen-activated protein kinase kinase). The Schiff base forming class of immunopotentiatory drug provides the first orally active, mechanism-based immunopotentiatory agents for therapeutic testing. Tucaresol is currently undergoing pilot phase I/II clinical trials as an immunopotentiator in chronic hepatitis B virus infection, HIV infection and malignant melanoma.     

Distinct regulation of T-cell death by CD28 depending on both its aggregation and T-cell receptor triggering: a role for Fas-FasL

CD28 is a major coreceptor that regulates cell proliferation, anergy, and viability of T cells. The negative selection by T-cell receptor (TCR)-induced cell death of immature thymocytes as well as of activated human antigen-specific T-cell clone, requires a costimulatory signal that can be provided by CD28. Conversely, CD28-mediated signals increase expression of Bcl-XL, a survival gene, and promote survival of naive T cells cultured in the absence of antigen or costimulation. Because CD28 appears to both protect from, or induce T-cell death, one important question is to define the activation and cellular parameters that dictate the differential role of CD28 in T-cell apoptosis. Here, we compared different CD28 ligands for their ability to regulate TCR-induced cell death of a murine T-cell hybridoma. In these cells, TCR triggering induced expression of Fas and FasL, and cell death was prevented by anti-Fas blocking monoclonal antibody (MoAb). When provided as a costimulus, both CD28 MoAb and the B7.1 and B7.2 counter receptors downregulated, yet did not completely abolish T-cell receptor-induced apoptosis. This CD28 cosignal resulted in both upregulation of Bcl-XL and prevention of FasL expression. In marked contrast, when given as a single signal, CD28 MoAb or B7.1 and B7.2 induced FasL expression and resulted in T-cell death by apoptosis, which was dependent on the level of CD28 ligation. Furthermore, triggering of CD28 upregulated FasL and induced a marked T-cell death of previously activated normal peripheral T cells. Our results identify Fas and FasL as crucial targets of CD28 in T-cell death regulation and show that within the same cell population, depending on its engagement as a single signal or as a costimulus together with the TCR, CD28 can either induce a dose-dependent death signal or protect from cell death, respectively. These data provide important insights into the role of CD28 in T-cell homeostasis and its possible implication in neoplastic disorders.     

B7-mediated costimulation can either provoke or prevent clinical manifestations of experimental allergic encephalomyelitis

T-cell activation requires signalling provided by ligation of the T-cell receptor for antigen (TCR) and a second antigen (Ag) nonspecific signal, known as costimulation. The B7 receptors, CD80 (B7-1) and CD86 (B7-2), on the Ag-presenting cell (APC), interact with T-cell CD28 or CTLA-4 to deliver a costimulatory signal, which is particularly important for Th1 activation. Experimental allergic encephalomyelitis (EAE) is an autoimmune disorder, induced by Th1 cells directed against myelin antigens that provides an in vivo model for studying the role of B7-mediated costimulation in the induction of a pathological immune response. Using a soluble fusion protein ligand for the B7 receptors, as well as specific monoclonal antibodies specific for either CD80 or CD86, it has been demonstrated that B7 costimulation plays a prominent role in determining clinical disease outcome in EAE. Here we review recent data indicating that a paradoxical exacerbation of disease as well as the expected amelioration of disease can occur with costimulatory receptor blockade.     

Compensation for decreased expression of B7 molecules on Leishmania infantum-infected canine macrophages results in restoration of parasite-specific T-cell proliferation and gamma interferon production

Infection of humans and dogs by Leishmania infantum may result in visceral leishmaniasis, which is characterized by impaired T-cell-mediated immune responses to parasite antigens. Dogs are natural hosts of Leishmania parasites and play an important role in the transmission of the parasites to humans. In an effort to characterize the immune response in dogs infected with this intracellular pathogen, we examined how infection with L. infantum affects canine macrophages and the consequences for T-cell activation in vitro. We showed that the proliferation of T-cell lines to cognate antigen decreases to background levels when infected autologous monocyte-derived macrophages are used as antigen-presenting cells (APC). The observed reduction of antigen-specific T-cell proliferation was shown to be dependent on the parasite load and to require cell-to-cell interaction of T cells with the infected APC. In addition, we observed a decreased expression of costimulatory B7 molecules on infected monocyte-derived macrophages. The expression of other surface molecules involved in T-cell activation, such as major histocompatibility complex class I and class II, on these cells did not change upon infection, whereas the expression of intracellular adhesion molecule 1 was marginally increased. Compensation for the decreased expression of B7 molecules by the addition of B7-transfected cells resulted in the restoration of cell proliferation and gamma interferon (IFN-gamma) production by a Leishmania-specific T-cell line. These results showed that for the activation of parasite-specific canine T cells producing IFN-gamma, which are most likely involved in protective immunity, sufficient expression of B7 molecules on infected macrophages is required. Provision of costimulatory molecules may be an approach for immunotherapy of leishmaniaisis as well as for vaccine development.     

Total hip arthroplasty: imaging evaluation

Crosslinking of CD28 receptors on resting T lymphocytes by B7 costimulatory molecules expressed by antigen-presenting cells (APCs) plays a critical role in T-cell activation. Human melanomas express major histocompatibility complex (MHC)-restricted tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTL), yet they remain poorly immunogenic. One mechanism for the failure of T-cell response is the lack of expression of costimulatory molecules by human melanoma cells. We have transfected the B7-1 gene into three HLA-A2-expressing human melanoma cell lines, and studied their capacity to stimulate primary human T cells. B7-expressing melanoma cells were excellent inducers of T-cell proliferation, cytokine production, and cytolytic activity in allogeneic mixed lymphocyte cultures through a process dependent on the function of the T-cell receptor as well as interactions between B7:CD28, CD2:LFA-3, and LFA-1:ICAM-1. Subset analysis demonstrated that CD4+ T cells or addition of exogenous interleukin-2 was required for the induction of CD8+ CTL. Untransfected parental melanoma cells were inert as APCs in these cultures. Rotating stimulation of T cells with the three B7-expressing cell lines led to the generation of T-cell lines that were cytolytic for HLA-A2+ melanoma cells and other HLA-A2+ targets that were pulsed with HLA-A2-restricted MART-1 peptides. These data demonstrate that expression of B7-1 by human melanoma cells converts them into effective APCs for the in vitro induction of MHC-restricted, melanoma-specific CTL.     

Anticryptococcal activity by alveolar macrophages from rats treated with cortisone acetate during different periods of time

Recently mouse models have shown that expression of costimulatory molecules such as B7-1 on tumor cells can induce tumor-specific immunity, suggesting that tumor cells modified to express costimulatory molecules can be a potential tumor vaccine. To investigate the importance of B7-1 co-stimulation in induction of autologous tumor immunity in humans, we established a renal carcinoma cell line, RCC-1, from a tumor resection and studied the patient's antitumor immune responses in vitro. The RCC-1 cell line constitutively expressed major histocompatibility complex (MHC) class I, intercellular adhesion molecule (ICAM)-1, and leukocyte function-associated antigen (LFA)-3 molecules, and MHC class II molecules were induced by interferon-gamma (IFN-gamma) treatment in vitro. However, neither RCC-1- nor IFN-gamma-treated RCC-1 cells expressed B7-1, and both failed to induce T-cell proliferative responses in mixed lymphocyte and tumor cell reaction (MLTR) assays, suggesting that the costimulatory signals provided by cell adhesion molecules such as ICAM-1 and LFA-3 were not sufficient to elicit an antitumor immune response. However, on transfection of the human B7-1 into RCC-1, these cells were able to induce a significant T-cell proliferation in MLTR assays. This T-cell response could be blocked by anti-B7 mAb treatment of the tumor cells. RCC-1B7 cells also induced the generation of tumor-specific cytolytic T lymphocytes to the parent RCC-1 cells in vitro, with little nonspecific cytolysis of an unrelated RCC line, A498, or autologous phytohemagglutinin (PHA) blasts. This specific cytotoxicity could be abrogated by anti-CD8 mAb and complement treatment. In summary, our study indicates that B7-1-CD28 interaction plays a critical role in induction of autologous tumor-specific cytotoxic T lymphocytes (CTLs) in humans, suggesting that the costimulatory molecule transfected tumor cells could be useful in expanding tumor-specific autologous CTL in vitro for adoptive tumor immunotherapy.     

Impaired categorical perception of synthetic speech sounds in schizophrenia

BACKGROUND: The rationale for the study was based on the hypothesis that decreased or absent expression on tumor cells of adhesion molecules, the class I or class II major histocompatibility complex (MHC) molecules, or costimulatory molecules might be responsible, in part, for the poor ability of squamous cell carcinoma of the head and neck (SCCHN) to induce generation of antitumor effector cells in vitro and in vivo. OBJECTIVE: To investigate expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-3 (LFA-3) and distribution of the costimulatory molecules, B7.1, B7.2, and CD40, and of class I and class II MHC molecules on SCCHN cells in situ and on SCCHN cell lines. SETTING: University medical centers. DESIGN: Expression of ICAM-1, LFA-3, MHC molecules, B7.1, B7.2, and CD40 was evaluated in human SCCHN biopsy specimens by immunohistochemistry and on SCCHN cell lines by flow cytometry. To confirm our hypothesis that impaired T-cell activation observed in patients with SCCHN is caused by the absence of costimulatory B7 molecules, a B7-negative SCCHN cell line was transduced with the B7.1 gene, using a retroviral vector, and tested in mixed lymphocyte tumor cocultures. RESULTS: In contrast to abundant expression of ICAM-1, LFA-3, class I MHC molecules, and CD40, the absence of B7.1, B7.2, and class II MHC molecules on tumor cells was observed in situ and in vitro. Lymphocytes and antigen-presenting cells in inflammatory infiltrates surrounding tumor cell clusters expressed both costimulatory and adhesion molecules. The SCCHN lines negative for B7.1 and class II MHC antigens failed to induce proliferation of T cells in mixed lymphocyte tumor cocultures. However, when these cell lines were transduced with the B7.1 gene, their ability to induce T-cell proliferation in mixed lymphocyte tumor cocultures was restored. CONCLUSIONS: The absence of B7 protein or class II MHC antigen expression on human SCCHN cells is responsible for the failure of these tumors to induce proliferation of T cells in vitro. Transduction of the B7.1 gene into SCCHN restores the ability of the tumor to induce T-cell proliferation in vitro.     

Identification of T-cell epitopes using allele-specific ligand motifs

In order to facilitate the identification of T-cell epitopes as useful components of synthetic vaccines, we investigated the role of MHC molecules as the restriction element for the recognition of epitopes by the alpha beta receptor of T cells. MHC molecules are able to present thousands of different peptides to T cells, with all the peptides presented by one distinct type of MHC sharing common structural features. Our group analyzed these common characteristics concerning peptide length (only MHC I ligands) and anchor positions (MHC I and II ligands) occupied by a small set of closely related amino acids. Until now, for more than fifty MHC proteins allele-specific "peptide motifs" have been defined. The exact knowledge of MHC I peptide motifs allows for a prediction of CTL epitopes, and this kind of prediction has been successful in many cases over the last three years.     

Costimulation provided by DNA immunization enhances antitumor immunity

The interaction of the TCR with MHC class I-bound Ag is insufficient for the priming of CTL unless secondary costimulatory signals are provided. To ascertain the minimum elements required to activate an Ag-specific CTL response in vivo, we injected mice intradermally or i.m. with plasmid DNA encoding a MHC class I-restricted peptide Ag (minigene) and different membrane-bound costimulatory ligands. The minigene-encoded epitope only primed a specific CTL response if injected in the vicinity of an ectopically expressed costimulatory ligand. Vector encoding B7-1 was repeatedly more potent at stimulating a cytolytic response than vector encoding B7-2. In contrast the B7-2-encoding plasmid preferentially enhanced Ag-specific Ab responses when injected with either protein or a cDNA expression vector. Gene vaccination with plasmids encoding OVA and B7-1, but not B7-2, prolonged survival in mice challenged with an OVA-transfected tumor. These results show that functional B7-1 transfection can be achieved in vivo and induces the selective induction of CTL. The data suggest that B7-1 plasmids should be coadministered with naked DNA vaccines that aim to induce tumor-specific cellular immunity.     

Low T-cell responses to CD3 plus CD28 monoclonal antibodies are predictive of development of AIDS

OBJECTIVE: Decreased T-cell reactivity in vitro is strongly associated with progression to AIDS and low CD4+ T-cell numbers. Low T-cell responses in vitro induced by CD3 monoclonal antibody (mAb) are predictive for progression to AIDS independent of low CD4+ T-cell counts and high HIV-1 RNA levels. We developed a whole-blood lymphocyte culture system in which T cells were stimulated by a combination of CD3 and CD28 mAb. Combined stimulation of CD28, a costimulatory molecule, and CD3 considerably enhances T-cell responses in vitro and reduces variation coefficients, which may increase the prognostic power of T-cell responses. DESIGN: A prospective study of HIV-1-infected homosexual men followed for 35 months. METHODS: The predictive value of low T-cell responses to CD3 plus CD28 mAb relative to low CD4+ T-cell counts, high HIV-1 RNA levels and the presence of syncytium-inducing (SI) HIV-1 variants was evaluated longitudinally in 202 HIV-1-infected homosexual men followed for 35 months. RESULTS: In multivariate analysis, decreased T-cell responses at baseline were predictive of development of AIDS, independent of low CD4+ T-cell numbers and high HIV-1 RNA levels. In a time-dependent model, HIV-1 RNA levels lost their predictive value, whereas low T-cell responses, low CD4+ T-cell numbers and the presence of SI HIV-1 variants independently predicted AIDS. CONCLUSIONS: These data demonstrate that combined use of virological and immunological markers may be useful in monitoring disease progression and response to antiretroviral therapy.     

Modification of human T-cell responses by altered peptide ligands: a new approach to antigen-specific modification

Human CD4+ T-cells recognize antigenic peptides in the context of human leukocyte antigen (HLA) class II molecules and produce various lymphokines to proliferate and activate other cells. It was once considered that the T-cell response is an all or nothing type event, but recent studies have clearly indicated that T-cells show many different types of activation in recognition of altered ligands for T-cell receptors (TCR). In this review, we summarize our recent findings on the human CD4+ T-cell response to altered peptide ligands (APL); peptides carrying single residue substitutions in antigenic peptides. We observed the following: 1) TCR antagonism for T-cell clones reactive to non-self or autoantigenic peptides, 2) partial activation (agonism) without cell proliferation, including production of lymphokines and increases in cell size, and in expression levels of several cell surface proteins or survival time in the absence of antigenic stimulus, 3) augmentation in cell proliferation and production of interferon-gamma (IFN-gamma) and granulocyte monocyte colony stimulating factor (GM-CSF), 4) augmentation of interleukin (IL)-12 production by antigen presenting cell (APC) and the subsequent augmented production of IFN-gamma by T-cells. This information provides basic knowledge regarding the characteristics of T-cell recognition of antigens and the subsequent activation, and a novel method for modification of human T-cell responses by altered peptide ligands (APLs), as a possible candidate for antigen-specific immunopotentiating or immunosuppressive therapy against autoimmune diseases, allergies, infectious diseases and malignant tumors.     

Myeloma bone marrow plasma cells: evidence for their capacity as antigen-presenting cells

Myeloma plasma cells constitute 10% to 90% of the total bone marrow cell count in patients with multiple myeloma (MM). These cells express a variety of cell surface markers, such as HLA-ABC and HLA-DR, and surface antigens that are necessary for professional antigen-presenting cells, including adhesion and costimulatory molecules. In this study, we examined the expression of major histocompatability complex (MHC) and costimulatory molecules on CD38(bright,++) plasma cells in bone marrow aspirates from eight MM patients. Small percentages of plasma cells expressed weak but detectable levels of HLA-DR, HLA-DQ, CD40, CD80, and CD86, which could be upregulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. CD38++ plasma cell and CD38(dim,+) cells were sorted from freshly isolated bone marrow mononuclear cells and tested for their capacity to act as antigen-presenting cells. Indeed, both CD38++ plasma cells and CD38+ cells were able to stimulate allogeneic T cells and present the soluble antigens purified protein derivative and tetanus toxoid to autologous T cells. Recognition of the antigens led to T-cell proliferation and secretion of IFN-gamma and was MHC class-I and -II restricted. Antigen processing and presentation by CD38++ and CD38+ cells were abolished by treatment of the cells with chloroquine. Hence, our study provides for the first time evidence that myeloma plasma cells may act as antigen-presenting cells. Further studies are warranted to examine in detail the molecules required for inducing T-cell stimulation.     

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes

Dendritic cells (DCs) are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses in vivo. Here, we show that DCs secrete antigen presenting vesicles, called exosomes, which express functional Major Histocompatibility Complex class I and class II, and T-cell costimulatory molecules. Tumor peptide-pulsed DC-derived exosomes prime specific cytotoxic T lymphocytes in vivo and eradicate or suppress growth of established murine tumors in a T cell-dependent manner. Exosome-based cell-free vaccines represent an alternative to DC adoptive therapy for suppressing tumor growth.     

Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association

Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).     

Eradication of pre-established lymphoma using herpes simplex virus amplicon vectors

Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing beta-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.