Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus.

Human monoclonal antibodies directed against human cytomegalovirus were generated by fusion of in vitro stimulated human spleen lymphocytes from an HCMV-seropositive 53-year-old organ donor with the mouse myeloma cell line Ag8.653. Fourteen human/mouse hybridomas producing anti-cytomegalovirus IgG were screened by an ELISA technique and four selected clones have been established since March 1988, generating about 5-40 micrograms/24 h IgG per ml culture supernatant. Reference and local cytomegalovirus strains were stained by the antibodies without showing cross-reactivity to other herpes viruses. Three monoclonal antibodies, A4B4 (IgG11), A6B3 (IgG1k) and A6A2 (IgG1k), immunoprecipitated a 68 kDa early viral protein which appears during the infectious cycle, first in the nucleus (18-24 h) and then also in the cytoplasm (24-96 h) of infected cells. Inhibition of DNA replication restricted the detection of the 68 kDa viral protein to the nucleus of infected cells. Staining of unfixed infected cells showed that two of the antibodies bound at the surface of a few cells. The fourth monoclonal antibody A3C5 (IgG11) immunoprecipitated a 34/38 kDa late viral protein which appears in the nucleus (48-72 h) of infected cells. These antibodies enable us to study the human host response to human cytomegalovirus and to elucidate the functions of human antibodies especially in their interaction with the T-cell response.

Coexistence of donor and host T lymphocytes following HLA-different bone marrow transplantation into a patient with cellular immunodeficiency and nonfunctional CD4+ T cells.

We report the outcome of a non-T-cell-depleted bone marrow transplant from an HLA partially incompatible, MLR-positive, parental donor in a patient with an unusual form of immunodeficiency characterized by a lack of CD8 T cells and a failure of the CD4 cells to display functional activity in vitro. Without conditioning, and following a mild and transient GVHD, donor T cells persist in trace amounts in the host, where they coexist with the nonfunctional host T cells and cooperate with host APC in antigen recognition, thereby leading to a reconstitution of T cell functions in vitro and in vivo and development of a stable, so far unprecedented, human T-T split chimera across MHC barriers.

Expression of MHC class I and II molecules by cadaver retinal pigment epithelium cells: optimization of post-mortem HLA typing.

The objective of this study was to investigate the expression of MHC antigens by retinal pigment epithelium cells (RPE) after stimulation with interferon-gamma (IFN-gamma) and to improve the currently practised technique of cadaver HLA typing. A concentration of 100 U/ml IFN-gamma induced expression of class I molecules up to greater than 90% 3 days after stimulation, whereas 50 U/ml were required for the expression of HLA-DR to greater than 90%. A concentration of 750 U/ml induced 35-45% expression of HLA-DP and less than 25% HLA-DQ after 3 days. Cells were serologically typed using the standard lymphocytotoxicity assay 3 days after stimulation with 250 U/ml IFN-gamma. Typing of class I specificities was complemented by one-dimensional isoelectric focusing (1D-IEF). We observed high concordance between the results of the RPE typing and the lymphocytotoxicity test on the same donors. Our results show complete typing of class I and II antigens post-mortem, which, in particular, enables graft matching and improvement of graft survival in recipients of organs removed many hours after death such as the cornea.

Human monoclonal antibody Ha6D3, a candidate for treatment of leukaemia? In vitro reactivity of Ha6D3 with leukaemic cells and in vivo applications in a chimpanzee.

The human monoclonal antibody Ha6D3 of the IgM type was used to stain malignant lymphoma cells from peripheral blood in flow cytometry and from cryosections of lymph nodes using the immunoperoxidase technique. It was found to react with peripheral white blood cells of all 12 cases of leukaemia and with lymph node cells of seven out of 11 B cell lymphomas and with the one T cell lymphoma tested so far. For in vivo experiments a batch of 70 mg Ha6D3 was purified and 6 mg Ha6D3 was injected intravenously into a chimpanzee with time intervals of 10 months and 1 month. The side effects observed were shivering, some muscular spasms and variations in the heart frequency. A decrease of lymphocytes of more than 50% was documented by haematogram analysis. The flow cytometry data showed that the Ha6D3 antigen does not modulate. Even after three repeated injections applied in a time interval of several months no immune response to Ha6D3 could be detected in vivo or in vitro. Based on these data we suggest that Ha6D3 may become a candidate for the treatment of certain leukaemias in vivo.

Serological mapping of HLA-epitopes with monoclonal antibodies and its interpretation by sequenced HLA-molecules.

For the study of the epitope distribution on HLA class I molecules, blocking studies were carried out with a competitive radioimmuno assay using 3H labelled monoclonal antibodies (MAbs) against various HLA epitopes. The results were discussed with the help of known HLA amino acid sequences, and the positions of the following epitopes were suggested: A2/Aw69; A2/28; A2/11/25/26/28/29/30/31/Aw33/34; A25/32; Bw4; B7/Bw22,42; Bw6.

Immunoelectron microscopic demonstration of antigenic sites on lymphoid cells using a human monoclonal antibody (Ha6D3).

The reactivity of a human monoclonal antibody directed against human B and T lymphocytes was tested for the first time at the ultrastructural level. The antigenic sites detected by this antibody were localized on the surface membrane of lymphocytes and, to a lesser extent, in the cytoplasm on membranes of the endoplasmic reticulum and perinuclear envelop of some centroblasts and immunoblasts. Ultrastructural demonstration of target antigen detected by human monoclonal antibodies may be important prior to therapeutic application of these antibodies.

Pregnancy-maintaining antibodies: workshop report (Giessen, 1988).

To analyse the nature of antibodies which are purported to be essential for the maintenance of normal human pregnancy, six centers participated in a workshop of "blind" tests on 19 allosera. Fc-receptor dependent assays detected antibodies with specificity only for HLA. In addition to cytotoxic antibodies, the Fc-receptor dependent immune phagocytosis inhibition test revealed two non-cytotoxic alloantibodies with HLA specificity. These antibodies had high titers and may, therefore, be essentially non-cytotoxic. Murine monoclonal antibodies to HLA-A, B, C or DR (W6/32 and 2MC3) were used to evaluate the methods. These antibodies inhibited immune rosette formation as well as immune phagocytosis. Diluted to concentrations below the threshold of complement-dependent cytotoxicity, the monoclonal antibodies still inhibited the mixed lymphocyte reaction and the immune phagocytosis. A human monoclonal immunoglobulin M with specificity for monomorphic non-HLA lymphocyte antigens inhibited the mixed lymphocyte reaction. The immune rosette inhibition test exhibited several false positive reactions, e.g. three out of four with a serum that did not contain alloantibodies to blood cells. Non-cytotoxic antibodies were therefore rare in the selected sera of the workshop and they exhibited HLA specificity only. No participant was able to identify pregnancy-maintaining non-HLA-antibodies.

Selective loss of HLA-A or HLA-B antigen expression in colon carcinoma.

A panel of colorectal carcinomas has been examined immunohistologically with mAb specific for allodeterminants of HLA-A or HLA-B, and with mAb reactive with HLA-A,B,C framework determinants or beta 2-microglobulin. Out of 85 carcinomas, 5 cases were completely negative for all class I Ag in the tumor cell population. Fifteen cases exhibited a differential expression of HLA-A and HLA-B products in all tumor cells or in a subset. Such tumor cells showed strong staining with monomorphic HLA antibodies but failed to stain with certain allospecific mAb. Negativity of tumor cells was scored only when normal stromal cells within the tumor mass were clearly labeled, indicating expression of the particular allodeterminant(s) in the given tissue. Four carcinomas showed a selective loss of HLA-A2 or a non-A2 HLA-A allospecificity in all tumor cells or a major subset, whereas HLA-B Ag were expressed. Seven cases showed a selective loss of the HLA-Bw6 superspecificity in the tumor cell population; HLA-Bw4 and HLA-A Ag were present. Three cases exhibited a combined loss of HLA-A2 and HLA-Bw6 Ag, with non-A2 HLA-A and HLA-Bw4 Ag being expressed. A further case was HLA-Bw6/Bw4 negative in a major tumor cell subset and HLA-A negative in the complementary minor subset. The results show that a considerable proportion of colorectal carcinomas is heterogeneous with regard to HLA-A and HLA-B expression. The reasons for the appearance of tumor subsets with complete loss of class I Ag or selective loss of only HLA-A or HLA-B Ag are not clear, but it is conceivable that some of them arose because they have escaped immunoselection.

Production and applications of new monoclonal antibodies against human lymphocyte antigen-A and -B antigens.

Monoclonal antibodies (MAbs) against HLA antigens can give better information about the serology and biochemistry of the human major histocompatibility complex (MHC) than HLA antisera obtained from pregnant women. To increase the very limited panel of MAbs against HLA we immunized mice with human cultured cell lines and fused their spleen cells with the Ag8-653 myeloma. We produced MAbs against HLA-A1, A2/w69, A2/28, A2/11/25/26/28/29/30/31/33/34, A25/32, B7/22, and B13. The best dilution medium to store the MAbs in Terasaki plates was RPMI 1640 supplemented with 7% bovine serum albumin. The MAbs are also excellent reagents to investigate public HLA antigens and to stain HLA antigens in cryosections of transplanted organs.

Human monoclonal antibody against human lymphocytic cells. A human monoclonal antibody that reacts preferentially with human lymphocytic cells.

Human monoclonal antibodies may replace human or xenogeneic antisera and mouse monoclonal antibodies for therapeutic applications. The human IgM monoclonal antibody Ha6D3 was produced after in vitro immunization and fusion with a mouse myeloma. Its reactivity against human normal and leukemic cells was investigated in cytotoxicity assays, and the antigen distribution in normal tissues was investigated with biotinylated Ha6D3 and avidin-peroxidase complexes. It was shown that Ha6D3 reacts preferentially with human lymphocytic cells. Only moderate reactions with epithelial cells in some organs were observed in cryostat sections, but because of poor accessibility in vivo these reactions were considered to be negligible.

Immunohistochemical characterization of the thymic microenvironment. A light-microscopic and ultrastructural immunocytochemical study.

The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells. The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles. The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.