The TAPA-1 molecule is associated on the surface of B cells with HLA-DR molecules.

TAPA-1 is a transmembrane protein that has been shown to be involved in cell growth and cellular adhesion. Our studies were aimed at determining the mechanisms of the biologic phenomena mediated by TAPA-1, which include the identification of proteins that are associated with it on the surface of lymphocytes. We and others have previously shown that Leu-13, a leukocyte Ag, is one such molecule and that in B cells TAPA-1 is associated with the CD19 Ag. Herein we identify an additional molecule, HLA-DR, that is noncovalently associated on the surface of B cells with TAPA-1. This association was first detected by immunoprecipitation by anti-TAPA-1 and by anti-HLA-DR antibodies in the presence of mild detergents. The initial observation was confirmed by 2-dimensional SDS-PAGE and by direct identification of TAPA-1 in anti-HLA-DR immunoprecipitates by Western blot analysis. The association of the two molecules on the surface of a human B cell line was shown by cocapping experiments. In addition, antibodies to both molecules can induce cellular adhesion and an antiproliferative effect. Because the tissue distribution of these two molecules only partially overlaps, with TAPA-1 being expressed on most cell types and MHC class II expressed on a more restricted group of tissue, it is possible that the TAPA-1 molecule provides a basic function that can augment a cell type specific activity. In B cells the association of TAPA-1 with CD19 and HLA-DR may increase cellular interaction and play a supporting role in the transmission of specific signals.

Anti-TAPA-1 antibodies induce protein tyrosine phosphorylation that is prevented by increasing intracellular thiol levels.

We studied the signal induced by the anti-TAPA-1 antibody and compared it to the signal induced by anti-IgM antibodies in a human B cell line, OCl-LY8. We found that exposure of these cells to either antibody resulted in a rapid increase in protein tyrosine phosphorylation which was prevented by inhibitors of tyrosine kinases. Tyrosine phosphorylation was an early event in the cascase leading to the antiproliferative effect of the anti-TAPA-1 antibody. However, 2-ME, a reducing agent that is not an inhibitor of tyrosine kinases, prevented both tyrosine phosphorylation and the antiproliferative effect of the antibody. Cells grown in low concentrations of 2-ME did not exhibit an increase in tyrosine phosphorylation in response to the anti-TAPA-1 antibody and were insensitive to the antiproliferative effect of the antibody. In contrast, the same cells maintained in 2-ME were able to induce tyrosine phosphorylation in response to anti-IgM. The use of 2-ME resulted in an increase in intracellular thiols, mostly glutathione. Moreover, compounds that block glutathione synthesis rendered cells susceptible to the antibody, even in the presence of 2-ME. These experiments demonstrate that tyrosine kinases are involved in propagating the antiproliferative signal initiated by the anti-TAPA-1 antibody and suggest that this signal is dependent upon the level of intracellular thiols.

Human T-cell development in SCID-hu mice: staphylococcal enterotoxins induce specific clonal deletions, proliferation, and anergy.

SCID-hu mice provide an in vivo model for studying the events of normal intrathymic human T-cell development and differentiation. We injected SCID-hu mice with staphylococcal enterotoxins (SE) and determined their effects on the development and responsiveness of human T-cell populations defined by their expression of CD4 and CD8, and the type of V beta molecule in their T-cell receptors. After single intraperitoneal injections of SEB or SEE, we observed specific effects on thymic T cells expressing a cognate V beta T-cell receptor (TCR) (V beta 12.1 in the case of SEB-treated SCID-hu mice and V beta 8.1 in the case of SEE-treated mice) using both immunohistochemical staining of thymic frozen sections and flow cytometric analyses. An injection of SEB resulted in a 32% decrease in the total percentages of V beta 12.1+ cells in thymic sections after 2 days, with the greatest effect seen in the medulla, without a demonstrable effect on V beta 5.2/5.3+ or V beta 8.1+ cells. Fluorescence-activated cell sorter analysis demonstrated that TCRhi thymocytes expressing a cognate V beta TCR declined transiently by 35% to 45% 1 to 2 days after the injection of SE. Analysis of thymic subpopulations showed decreases in the TCRhi CD4+8- and CD4-8+ cells and an increase in TCRlo CD4-8+ cells. Multiple injections of SE resulted in 50% to 60% decreases in cognate V beta TCR+ CD4+8- populations. Thymocytes prepared from SE-treated SCID-hu mice demonstrated specific anergy to the SE to which they had previously been exposed in vivo, but had a normal proliferative response to other superantigens in an in vitro assay. In contrast to the effects on thymic T cells, single injections of SE resulted in a twofold increase in the total numbers of circulating CD4+8- and CD4-8+ human T cells and a fourfold to eightfold increase in T cells expressing a cognate V beta TCR. Using SE as superantigens in SCID-hu mice, we have been able to induce antigen-specific clonal deletions, anergy, and proliferation of human T cells.

Growth of primary T-cell non-Hodgkin's lymphomata in SCID-hu mice: requirement for a human lymphoid microenvironment.

We reasoned that the SCID-hu mouse could provide an appropriate lymphoid or stromal microenvironment to support the growth of primary human lymphoma. Heterotransplantation of nine cases of primary T-cell non-Hodgkin's lymphoma (NHL) into untreated SCID mice and SCID mice reconstituted with human fetal thymus, spleen, and liver (SCID-hu) resulted in the development of lymphoid tumors in five (56%) cases. Two clonal T-cell NHL grew after a mean of 90 days after injection of primary lymphoma cell suspensions into the thymus xenografts in SCID-hu mice and failed to grow in a variety of sites in SCID mice, except for small tumors that developed after a long (157-day) latency period after intracranial injection of tumor cell suspensions into weanling SCID mice. Successful serial transplantation of NHL in SCID and SCID-hu mice required the presence of a human lymphoid or tumor microenvironment, and was enhanced by pretreating the SCID mice with 175 rad radiation and antiasialo antisera. Analysis of the primary and transplanted T-cell tumors showed identical patterns of T-cell surface markers by flow cytometry and immunophenotyping of fixed tissue sections, and, in one case, reactivity with a specific monoclonal antibody to V beta 5.1. Genotyping of the transplanted tumors showed T-cell receptor gene rearrangements identical to those present in the primary tumors. In one case, the presence of Epstein-Barr virus-positive B cells in association with the primary tumor resulted in the growth of a lymphoblastoid B-cell neoplasm in addition to the malignant T-cell lymphoma after transplantation of tumor fragments to SCID mice. The data support the hypothesis that a human lymphoid microenvironment enhances the growth of T-cell NHL in SCID mice. The SCID-hu thymus graft provides an apparently unique microenvironment that supports the growth of primary T-cell NHL, and can be used to study the interaction between lymphoma cells, nontransformed lymphoid cells, and the surrounding stromal microenvironment in vivo.

Anti-idiotypic antibodies against a monoclonal antibody specific for the trichothecene mycotoxin T-2.

A BALB/c murine monoclonal antibody against the trichothecene mycotoxin T-2 was generated. The antibody, designated HD11, specifically bound T-2 mycotoxin. The binding of HD11 to T-2 conjugated to bovine serum albumin was inhibited by free T-2 toxin but not by the water-soluble heterocyclic guanidines saxitoxin and tetrodotoxin. The T-2 detection limit in an enzyme-linked immunosorbent assay with HD11 was in the nanogram range. The in vitro cytotoxicity of T-2, as measured by the inhibition of radiolabeled leucine uptake of the human epidermoid carcinoma Hep-2 and KB cell lines, was completely reversed by the addition of HD11. Rabbit anti-idiotypic antibodies specific for HD11 were generated and characterized.

Methods for generating reagents to examine idiotype networks within antiviral immune responses.

The use of anti-idiotypic antibodies to examine and/or modulate the immune response to various viral antigens has the potential to be of use in many diverse systems. This paper details the method and immunologic parameters used in our laboratory to generate and characterize anti-idiotypic antibodies (anti-Id or Ab-2) with specificity for antibodies directed against viral antigens. These anti-Id reagents have been used in our laboratory for studies involving the immune responses to hepatitis B virus and simian virus 40, which we describe here, as well as herpes simplex virus, and the human immunodeficiency virus. We have utilized these anti-Id reagents to examine the fine specificity of the idiotypes on antiviral antibodies in these systems and have attempted to modulate or induce specific antiviral immune responses. It is anticipated that the methods described herein will be helpful in analyzing the immune response in other viral systems including studies involving viral-receptor interactions.

Induction of an anti-hepatitis B surface antigen response in mice by noninternal image (Ab2 alpha) anti-idiotypic antibodies.

Anti-idiotype antibodies to a mouse monoclonal antibody A-12 directed against HBsAg were produced in rabbits. The anti-Id consisted of an Ab-2 alpha preparation that did not display any detectable internal image activity. Immunization of BALB/c mice with the anti-Id (Ab-2 alpha) coupled to KLH induced an anti-HBs response without subsequent HBsAg exposure. No anti-HBs was detected in control groups of mice immunized with other rabbit anti-Id-KLH preparations. The anti-HBs containing sera from mice immunized with the Ab-2 alpha were able to inhibit the Id-anti-Id reaction, indicating that an Id-positive, anti-HBs response was induced. This idiotype is not normally expressed during the murine immune response to HBsAg and suggests that noninternal image anti-Id activates silent clones. This study, along with our previous results obtained with the use of internal image anti-Id, suggests that there is more than one Id network operational during the BALB/c murine immune response to HBsAg.

Prediction of transient temperature fields and cumulative tissue destruction for radio frequency heating of a tumor.

A therapeutic hyperthermia protocol using a radio frequency (rf) electrode placed adjacent to a bronchial wall tumor has been modeled using the finite element technique. Variable physical properties and variable blood perfusion have been assigned to the tumor and to the surrounding normal lung tissue. The Laplace equation was solved on a curvilinear grid for a single rf source electrode to determine the steady-state electric field, which in turn governs the energy deposition function. The heat generation in the tumor and in the lung tissue is then calculated from the energy deposition profile, and the bioheat equation is solved on the same finite element mesh to determine the transient temperature history. The temperatures are displayed as isothermal contours at designated times during the protocol and as temperature histories at selected points. In addition, an Arrhenius-type injury model has been implemented to predict thermally induced damage, from which equal total amounts of energy are deposited into the tissue using a constant power density for an appropriate time or using a cyclic heating pattern. The cyclic heating pattern consisted of a series of equal duration time periods during which the rf current source is alternately turned on and off (50% duty cycle). This study illustrates how a finite element model could be used to evaluate alternative protocols for heating a tumor of a specific geometry and to evaluate thermally induced damage to surrounding normal tissue.