Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family.

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.