Tissue Typing of Cells in Culture. III. HLA Antigens of Established Human Cell Lines. Attempts at Typing by the Mixed Hemadsorption Technique

Thirty established cell lines from solid human tumors were typed by the mixed hemadsorption technique with respect to the HLA-A and HLA-B and partly also the HLA-C series. All cultures exhibited unique antigenic patterns. The discriminatory potential was further increased very much taking into consideration one as yet undefined antigen series tentatively named Ek-1 to Ek-11. Sixteen cell lines not designated as HeLa cells but on various non-immunological indications suspected to be HeLa cell contaminants, all gave, with our present test, the same antigenic composition as HeLa cells or clones thereof. The mixed hemadsorption reaction is a highly sensitive and convenient method for immunological typing of cells in monolayer cultures. Some important implications of this are discussed.     

HLA-D Typing with Lymphoblastoid Cell Lines IV. Allelic Relationships

Responses of approximately 200 persons were measured by mixed leukocyte cultures against 41 lymphoblastoid cell lines which were thought to be homozygous for HLA--D (i.e. LCL-HTCs). These responses were standardized by computer analysis and the resultant Interaction Indices for each LCL-HTC were compared with those of every other LCL-HTC in a correlation matrix of 861 independent analyses. Only those analyses involving LCL-HTCs of defined HLA--D types are reported here. In general, these studies confirmed many allelic relationships of reputedly similar LCL-HTCs; however, several unexpected groups of correlations suggested that groups Dw1 and 3 overlap to form Groups "1a-3," "1b-3" and "true 3." The "1b-3" group also included Dw8. Segregation of the "1b-3" allelic group was also observed in a family study. These preliminary analyses suggest a working model for further investigation of HLA--D allelic relationships (Fig. 3). On the other hand, they may also be interpreted to suggest that "typing" with LCL-HTCs may be controlled by a separate HLA locus which is closely related to, but distinct from, HLA--D.     

Epstein-Barr Virus-Transformed B Cell Lines Present M. Leprae Antigens to T Cells

We have investigated whether or not Epstein-Barr virus-transformed lymphoblastoid B cell lines (EBV-BLCL) are able to present Mycobacterium leprae (M. leprae) to antigen-reactive T cell lines and clones. Such EBV-BLCL would provide us with a homogeneous and unlimited source of antigen-presenting cells. Antigen-triggered proliferation of T cells has been studied with co-cultures either with autologous or allogeneic irradiated EBV-BLCL. Our results show that EBV-BLCL are able to present M. leprae as efficiently as peripheral blood mononuclear cells, and that they also function in an HLA-DR-restricted fashion. Apart from their possible in vivo relevance, these results have important practical implications, in particular for the generation and study of M. leprae-reactive T-cell clones.     

Lymphoblastoid Cell Lines of Homozygous Typing Cells Used for Sensitization in PLT

Lymphoblastoid cell lines of homozygous typing cells were used as the sensitizing cells in MLC to prepare PLT cells. Results obtained using such cells against a panel of restimulating cells were compared to those obtained using regular PLT cells in which priming had been accomplished with normal peripheral blood lymphocytes. It appears that lymphoblastoid cell lines can be used for this purpose; the advantages of such an approach are given.     

Tissue Typing of Cells in Culture. II. Histocompatibility Typing of Diploid Fibroblastic Cultures by the Mixed Hemadsorption (MH) Test

Skin fibroblasts derived from persons who had been HLA-typed by conventional lymphocytotoxocity tests were subjected to HLA-typing by the mixed hemadsorption (MH) test. The agreement between the two methods was good, provided sera for the MH test were carefully selected. Minor discrepancies found in this study can probably be corrected by further serum selection. There was no difficulty in typing late passages of cultured diploid cells (22 doublings) and in fact it was easier to type a culture at the 33rd doubling than at the 11th. Thus no signs of disappearance of HLA-antigens were seen in these tests.     

Expression of Human Placental Cell Surface Antigens on Peripheral Blood Lymphocytes and Lymphoblastoid Cell Lines

The expression of human placental cell surface antigens was examined in cells of lymphoid origin, including peripheral blood lymphocytes and cultured lymphoblastoid cells of bone marrow or thymus derivation. A select group of this. defined set of surface antigens was detected on all three cell preparations. The most remarkable observation was the conspicuous absence of three subunits previously demonstrated to be present on all human cell surfaces examined to date. Antiserum directed against several placental components prevents adhesion and spreading of cell which grow attached to surfaces. These results suggest a role for these three glycoproteins in mediating cellular adhesion.     

Associative Control of the Immune Response to Cell Surface Antigens

The immune response to cell surface antigens is regulated by the activity of helper and suppressor cells, probably of TH and possibly of TCS types. For this to operate, recognition must take place of two or more antigenic determinants, which may be carried on the same (intramolecular help) or different (intermolecular, intrastructural help) molecules. Regulation of this type has been shown to operate in the response to the murine allo-antigens H-2K. H-2D, Thy-1, and H-minors.     

Associative Control of the Immune Response to Cell Surface Antigens

The immune response to cell surface antigens is regulated by the activity of helper and suppressor cells, probably of TH and possibly of TCS types. For this to operate, recognition must take place of two or more antigenic determinants, which may be carried on the same (intramolecular help) or different (intermolecular, intrastructural help) molecules. Regulation of this type has been shown to operate in the response to the murine allo-antigens H-2K. H-2D, Thy-1, and H-minors.     

抗人肿瘤细胞核单克隆抗体的制备及鉴定

根据Epstein等报告,肿瘤坏死部分的不溶性抗原可以作为放射免疫检测与治疗的靶部位。作者制备了抗人肿瘤细胞核单克隆抗体(McAbs),并对其性质进行了鉴定。放射性同位素标记的抗核McAds的初步生物分布试验表明,抗核McAds确可到达肿瘤坏死部位。     

Complement-Fixing Antigens in Lymphoid Cell Lines of American Origin

Lymphoid cell lines from Americans with infectious mononucleosis (Choate, EH IV, and OP), and from a normal American (Cassio), were tested for presence of herpes-like virus by electron microscopy (EM) and immunofluorescence (IF), and for complement-fixing (CF) antigens, using both American and African sera. Whereas earlier tests of seven African (Burkitt) lymphoma cell lines showed an absolute correlation between presence of herpes-like virus and CF reactivity with either African or American sera, the same was not true of the American cell lines. Herpes-like particles were found in the Cassio line by both EM and IF, and in a few cells of the OP line by IF, but not by EM. The virus was not found in the Choate or EH IV lines by either EM or IF. African sera from either normal individuals or patients with Burkitt lymphoma contained CF antibodies to extracts of Cassio and OP cells. Normal American sera contained CF antibodies to these extracts as well as to extracts of Choate cells. The EH IV cell line did not produce CF antigens detectable with either African or American sera. The data indicate that the CF antigens of the herpes virus-negative Choate cell line were serologically distinct from those in Burkitt lines. However, it is possible that the antigens from both sources are related to the presence of the genome of the herpes-like virus.     

Synthesis and Expression of Surface Antigens During the Cell Cycle

The expression of H–2 histocompatibility antigens, assayed by immune cytolysis, is minimal during the S period of the cell cycle. In order to investigate this phenomenon, we have measured the amount of H–2 antigens on P815Y cells at different stages of the cell cycle by two different techniques. The first involves the binding of [¹²⁵I] labelled antibody, the second the inhibition of immune cytolysis of indicator cells by bound and salt-extractable antigens.
Each method shows that the amount of H–2 antigens increases during the G₁ period and then remains constant. On the other hand, the fragility of cells assayed by cytolysis caused by detergents, hypotonicity or freeze thawing shows the same minimum in S as does the expression of H–2 measured by immune cytolysis.
It is concluded that cell surface antigens are inserted into the plasma membrane during G₁ and remain accessible to combination with antibody thereafter. Because of a decrease in cellular fragility during interphase, cytolytic techniques should be used with caution.     

Epstein Barr Virus Transformation of Peripheral Blood B Cells Secreting Antibodies Reactive with Cell Surface Antigens

The natural structuring of the immune system is responsible for the functional physiological state of the body. The development of natural autoantibodies involved in homeostasis relies on the ability to distinguish between exposed/masked and altered/non-altered self antigens. The objectives of this article were to address the relationships between antigen and autoantibodies against serum amyloid A (SAA), define SAA protein concentrations in 219 blood donor (BD) sera and determine their autoantibody levels and search for possible clinical associations with autoimmune and thrombotic diseases. Just recently, an increasing number of reports have indicated significantly decreased levels of autoantibodies against pro-inflammatory molecules, such as anti-TNF-alpha, anti-IL-6, or anti-CRP found in diseased conditions, as compared to healthy donors, or even to less severe disease conditions. In accord with this line of thought, our data indicate a predominant presence of anti-SAA autoantibodies in healthy BDs (above 95% as tested by the immunoblot analysis, n = 41). Using ELISA, high levels of anti-SAA antibodies were confirmed with a median OD = 0.996 for the BD group (n = 219). This suggests that anti-SAA antibodies might have a physiological role in homeostasis and/or the innate immune system and could actually be a part of the natural antibody repertoire. Significantly, lower median levels were found in patients with arterial thrombosis. Based on 219 BD sera, we could establish a new median value of 20 μg/ml for SAA antigen and a cut-off value of 114.7 μg/ml (97.5th percentile). Significantly, higher concentrations of SAA were observed for antiphospholipid syndrome, rheumatoid arthritic, and SLE patients.     

Demonstration of Ia Antigens on Certain Dendritic Cells and on a Novel Elongate Cell Found in Human Synovial Tissue

Adherent cells from dissociated human synovial tissue obtained at surgery contain two types of distinctive cells with one or more elongated branching processes that strongly express Ia antigens. One type of cell with Ia antigens is non-phagocytic and resembles the murine dendritic cell. It is primarily found in patients with rheumatoid arthritis and accounts for a considerable proportion of the identifiable cells with a stellate or dendritic morphology. The expression of Ia antigens progressively diminished in culture. The second type of novel cell with Ia antigens was highly elongate and fibroblastoid. It was readily obtained from patients with osteoarthritis. The cell was frequently characterized by a blunt-ended filopodium-like process at one pole of the cell, one or two tapering processes, and zones of microvilli, Evidence was obtained suggesting that this cell, which might otherwise be considered fibroblast-like, is in the mononuclear phagocyte lineage.     

β2-Microglobulin on the Cell Surface. Relationship to HL-A Antigens and the Mixed Leucocyte Culture Reaction

Fab'-fragments derived from antibodies against β₂-microglobulin will completely abolish the cytotoxic effect of antibodies against HL-A serological determinants. This is the result of the Fab'-fragments competing with the antibodies against the HL-A antigens for binding to the lymphocyte surface. Reciprocally, it is shown that Fab'-fragments against the HL-A alloantigenic structures impeded the binding of Fab'-fragments to cell surface bound β₂-microglobulin.
Fab'-fragments against β₂-microglobulin inhibit considerably the mixed leucocyte culture reaction (MLR). The main effect is on the responding cells, whereas when the stimulating cells are treated with Fab'-fragments against β₂-microglobulin they do not (or, at most, only slightly) change their characteristics in the MLR. Fab'-fragments against immunoglobulin light chains do not influence the MLR significantly, whereas Fab'-fragments against HL-A antigens depress the response.
Fab'-fragments against β₂-microglobulin do not interfere with the mitogenic stimulation of PHA, whereas Fab'-fragments from the antiserum used in this study against HL-A8 reduced the mitogenic effect of PHA considerably.     

Enzyme-induced Aggregation and Disaggregation of Tumor Cells via the Cell Surface Glycocalyx in Association with Deoxyribonucleic Acid

Serine proteases cause aggregation of the rat ascites tumor cell lines AH 130, AH 109A and YS in vitro , and the tumor cell aggregates are dissolved by treatment with DNase I. We previously demonstrated that these events played a critical role in the augmentation or reduction of experimental blood borne metastasis of these cell lines. In the present study, the ultrastructural features of this protease dependent aggregation were analysed. Transmission and scanning electron microscopy revealed that after the protease treatment each tumor cell was surrounded by a thin membranous (sleeve-like) structure. This sleeve like structure was stained with ruthenium red to an intensity similar to the cell surface of the control. Adjacent cells became attached to each other with microvilli via this fine structure. Immuno-electron microscopy revealed DNA antigen as dense patches on the sleeve-like structure or as faint and diffuse deposits on the outer surface of the cells by indirect immunoperoxidase staining using an anti-DNA monoclonal antibody. Both the sleeve like structure and immunopositive deposits disappeared after treatment with DNase I. Neither cell viability nor the normal ultra-structure of their organelles was influenced by the enzyme treatment. These results indicate that serine protease induced tumor cell aggregation is due to cellular contact via the sleeve-like structure, which probably originates from the cell surface glycocalyx in association with DNA molecules of unknown origin.     

HLA—D Typing with Lymphoblastoid Cell Lines. VI. Rationale and Goals of Data Reduction

This report documents various characteristics of HLA-D typing by mixed leukocyte culture reactions when lymphoblastoid cell lines (LCLs) are substituted for peripheral blood lymphocytes as the stimulator cells. It also provides the rationale for designing the computer program described in the subsequent report. In such experiments, each donor to be HLA-D typed is stimulated with a panel of 30-50 HLA-D homozygous LCLs, each defined HLA-D allele being represented by several different human homogygous typing cells (LCT-HTCs). Variability in the strength of eahc donor's general response and in the strength of stimulation by each LCL-HTC makes it necessary to normalize raw data before the responses of various combinations can be compared and typing responses distinguished from non-typing responses. The autologous response and its equivalent effect among allogenetic combinations, the so-called "autologous-stimulation" effect, must also be distinguished from true allogeneic responses. The latter has been accomplished by "modelling," as described in the subsequent report. EBV-negative donors can also be HLA-D typed by this method despite the EBV-positivity of the LCL-HTCs. Preliminary analyses suggest that the HLA-D alleles defined by this method appear to segregate with appropriate haplotypes in family studies.     

Separation of Cells According to Surface Antigens by the Use of Antibody-Coated Columns. Fractionation of Cells Carrying Immunoglobulins and Blood Group Antigen

It has been found possible to separate cells according to their surface anti gens by the use of antibody-coated columns. High efficiency columns were made by double-layer principles, first coating beads with antigen followed by antibody in excess. Such columns could be shown to contain a high amount of free antigen-binding sites for the relevant antigens. Lymphoid cells were thus fractionated according to their surface concentration of immunoglobu lin and a highly selective retention of mouse B lymphocytes was observed when filtering spleen cells through an anti-immunoglobulin column prepared according to the above procedure. No evidence of retention of mouse T lymphoid cells was observed in the same system. By the use of anti-gamma-2a immunoglobulin columns, it was found possible to deplete a population from memory cells potentially capable of synthesizing gamma-2a antibodies. No evidence was found that columns prepared in the described manner would function through combining with receptors on lymphoid cells for antibody-antigen complexes. By using anti-A blood group columns, it was possible to selectively retain cells (erythrocytes or kidney cells) with A blood group anti gen on their surface. High-avidity immune antibodies were found to be more efficient than 'natural' anti-A antibodies in this test. No evidence was found of anti-A antibodies being adsorbed on to the passing cells as tested by in vitro serological tests and tissue culture experiments. The applications of a technique for separating cells according to their surface antigens are con sidered obvious.