Retroviral transfer of T-cell receptor genes produces cells with a broad range of lytic activity.

The ability to transfer the T-cell receptor (TCR) for antigen using a retroviral vector has opened the door to a new paradigm for T-cell-based immunotherapy. Using recombinant TCRs, a population of activated T cells can now be redirected to recognize and lyse cellular targets according to the specificity afforded by the transduced TCR genes. To examine the range of lytic activity displayed by the transduced TCRs, transduced T cells were re-cloned by limiting dilution and quantitatively analysed for lytic activity. The lytic activity of the transduced TCRs varied considerably, as determined by the Km and Vmax of lysis. The lytic activity seen in the secondary clones generated from vector-transduced peripheral blood mononuclear cell demonstrated that one of the clones approached the lytic activity of the parental 'TCR donor' cytotoxic T-cell lymphocyte (CTL) clone, whereas the remainder demonstrated either reduced Vmax or reduced Vmax and Km. Thus, the lytic activity of a transduced TCR depends not only on its genetic sequence but also on the cellular context within which it is expressed. Analysis of TCR Vbeta transcript levels by real time polymerase chain reaction revealed that while total Vbeta gene expression was fairly constant, expression of the retrovirally transduced Vbeta chain varied greatly in transduced CD8+ CTL clones.

Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine.

The absence of surface costimulatory molecules explains in part the lack of an effective anti-tumor immune response in tumor-bearing animals, even though unique tumor antigens may be presented by class I MHC. We determined that the immunogenicity of a murine neuroblastoma, Neuro-2a, which lacks surface costimulatory molecules, could be increased by electrically induced fusion with dendritic cells. Electrofusion induced a higher level of cell fusion than polyethylene glycol, and tumor/dendritic cell heterokaryons expressed high levels of costimulatory molecules. While Neuro-2a was unable to induce the proliferation of syngeneic or allogeneic T cells in vitro, fused cells were able to induce T cell responses both in vitro and in vivo. When fused dendritic tumor cells were used as a cancer vaccine, immunized mice were significantly protected from challenge with Neuro-2a. We propose that electrofusion with patient-derived tumor and dendritic cells may provide a rapid means to produce patient-specific tumor vaccines.

Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide.

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.

Detection of Epstein-Barr virus EBER sequence in post-transplant lymphoma patients with DNA dendrimers.

The EBER RNAs are the most numerous viral transcripts in latently infected lymphocytes in healthy individuals and also in the tumor cells of Epstein Barr virus (EBV)-associated malignancies. A rise in EBV load in peripheral blood has been associated with the onset of post-transplant lymphoproliferative disease (PTLD) in immunocompromised patients. Treatment of PTLD with adoptive immunotherapy has made the rapid and accurate determination of EBV load essential. The relationship between EBV load and other EBV-associated malignancies, like Hodgkin's disease or AIDS-associated lymphoma, is unknown. In order to define viral load based on the number of EBV-infected cells in the peripheral blood, we developed a method which combines cellular dilution of peripheral blood mononuclear cells with the direct detection of EBER-1 RNA with DNA dendrimers. DNA dendrimers are large scaffolds of DNA which give at least a 500 1000-fold increase in detection of membrane bound nucleic acid over oilgonucleotide probes. The use of a novel class of these nucleic acid superstructures is described as a specific probe for EBER-1 detection. When two PTLD patients were analyzed for viral load with DNA dendrimers, at least one in 250000 peripheral blood mononuclear cells were shown to be infected with EBV.

Determination of Epstein-Barr virus (EBV) load by RT-PCR and cellular dilution.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus which establishes life-long latency in the B-lymphocytes of infected individuals. Epstein-Barr virus is associated with Hodgkin's disease, AIDS-associated lymphoma and post-transplant lymphoproliferative disease (PTLD). In PTLD, the onset of malignancy correlates with a rise in EBV load. The relationship between malignancy and EBV load in other EBV-associated malignancies is not known. Epstein-Bar virus latency is associated with the expression of a limited set of viral transcripts. The most numerous of these are the EBERs (Epstein-Barr early RNAs). The high copy number of the EBERs in each latently infected cell led the author to combine serial dilution of lymphocytes with reverse transcriptase polymerase chain reaction (RT-PCR) for EBER-1 as a means to rapidly quantitate EBV load. The highest viral load was seen in a bone marrow transplant patient, where one in 3906 lymphocytes harboured EBV. Elevated viral load was seen in two solid-organ transplant patients. Viral loads in healthy volunteers ranged from less than one in 1x10(6) to one in 6.25x10(4). Reverse transcriptase polymerase chain reaction for EBER-1 in serial lymphocyte dilutions should allow the relationship of EBV load and malignancy to be examined in a number of disease settings and should also provide a quantitative picture of the impact of anti-viral therapy.

Feasibility of cellular adoptive immunotherapy for Epstein-Barr virus-associated lymphomas using haploidentical donors.

Epstein-Barr virus (EBV)-associated lymphomas following bone marrow or solid organ transplantation are often sensitive to immunomodulatory therapies. These have included withdrawal or reduction in immunosuppressive therapy in the solid organ transplant setting and adoptive cellular therapies in the bone marrow transplant (BMT) setting. We describe a strategy for generating EBV-specific cytotoxic T cell therapy lines with substantial killing activity against haploidentical targets. Weekly stimulation of peripheral blood mononuclear cells (PBMCs) for 3 weeks with the irradiated cells of an autologous EBV-transformed B lymphoblastoid cell line (B-LCL), followed by stimulation in the presence of IL-2, yielded T cell lines that were cytolytic for haploidentical B-LCLs but did not lyse haploidentical targets not expressing EBV antigens.

Association of host cell surface adhesion receptors and other membrane proteins with HIV and SIV.

We have developed a MAb-based capture assay to study the association of host cell membrane proteins with HIV and SIV. Class I and II MHC proteins were found to be associated with HIV as previously described. In addition to these molecules a number of other host molecules were found to be acquired by HIV, including CD71, CD63, CD43, and CD8. We have demonstrated that the major leukocyte adhesion receptors LFA-1 (CD11A/CD18) and CD44 are also associated with HIV. The level of surface expression of host membrane proteins did not predict relative expression (capture efficiency) of the virus. The use of virus-susceptible indicator cells showed that the assay involved host membrane protein-mediated capture of infectious HIV and SIV particles. Our data indicate that HIV and SIV acquire a number of host membrane proteins including adhesion receptors and that this process may be nonrandom. The acquisition of host cell adhesion receptors by HIV and SIV could have profound effects on the biology of the viruses, including binding, infectivity, and tropism.

Anti-class II MHC antibody induces multinucleated giant cell formation from peripheral blood monocytes.

Multinucleated giant cells (MGCs) are an integral part of the host immune response to infectious disease and are seen in granulomas induced by pathogens and inorganic substances. We have developed a novel system for the production and study of MGCs: Peripheral blood monocytes, when cultured in the presence of anti-class II major histocompatibility complex monoclonal antibodies (MHC mAb's) and lymphocyte-conditioned medium form MGCs within 48 h. MGC formation was strictly dependent on the presence of anti-class II MHC mAb's and lymphocyte-conditioned medium. MGC formation was not induced by mAb's to other monocyte surface proteins. None of the previously identified macrophage fusion factors (calcitriol, interleukin 4, interferon-gamma) were able to substitute for the lymphocyte-conditioned medium in our assay; however, the conditioned medium could be replaced by the phorbol ester phorbol 12-myristate 13-acetate. We have also demonstrated that the induction of MGCs by anti-class II MHC antibody and phorbol ester requires protein kinase C activity, because MGC formation was totally inhibited by the protein kinase C inhibitors staurosporine and H-7. In analyzing the signal induced by anti-class II MHC mAb's we have demonstrated that cross-linking of the class II MHC antigens with intact mAb's, or with F(ab')2 fragments of anti-class II MHC mAb's and F(ab')2 fragments of rabbit antimouse (RAM) immunoglobulin G, produced an intracellular calcium rise. Furthermore, using the calcium channel blocker verapamil, it was demonstrated that calcium channel activity is necessary for MGC formation. These data support the view that MGC formation is a tightly regulated differentiative pathway of peripheral blood monocytes that is dependent on protein kinase C second messenger systems and involves an increase in intracellular calcium concentration.

Induction of CD4+ human cytolytic T cells specific for HIV-infected cells by a gp160 subunit vaccine.

Cytolytic T lymphocyte (CTL) responses were evaluated in humans immunized with recombinant human immunodeficiency virus type 1 (HIV) envelope glycoprotein gp160. Some vaccinees had gp160-specific CTLs that were shown by cloning to be CD4+. Although induced by exogenous antigen, most gp160-specific CTL clones also recognized gp160 synthesized endogenously in target cells. These clones lysed autologous CD4+ T lymphoblasts infected with HIV. Of particular interest were certain vaccine-induced clones that lysed HIV-infected cells, recognized gp160 from diverse HIV isolates, and did not participate in "innocent bystander" killing of noninfected CD4+ T cells that had bound gp120.

Involvement of a leukocyte adhesion receptor (LFA-1) in HIV-induced syncytium formation.

Cell fusion (syncytium formation) is a major cytopathic effect of infection by human immunodeficiency virus (HIV) and may also represent an important mechanism of CD4+ T-cell depletion in individuals infected with HIV. Syncytium formation requires the interaction of CD4 on the surface of uninfected cells with HIV envelope glycoprotein gp120 expressed on HIV-infected cells. However, several observations suggest that molecules other than CD4 play a role in HIV-induced cell fusion. The leukocyte adhesion receptor LFA-1 is involved in a broad range of leukocyte interactions mediated by diverse receptor-ligand systems including CD4-class II major histocompatibility complex (MHC) molecules. Possible mimicry of class II MHC molecules by gp120 in its interaction with CD4 prompted an examination of the role of LFA-1 in HIV-induced cell fusion. A monoclonal antibody against LFA-1 completely inhibited HIV-induced syncytium formation. The antibody did not block binding of gp120 to CD4. This demonstrates that a molecule other than CD4 is also involved in cell fusion mediated by HIV.