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.     

Tissue Typing of Cells in Culture. IV. Cell Surface Antigens of Tumor Cell Lines, not Obviously Belonging to the HLA-System

Studies on the HLA pattern of several human tumor cell lines by the mixed hemadsorption test and also studies on the discriminative patterns formed by a collection of sera on a number of cell lines and diploid cells have been reported elsewhere. It was noted during these studies that some sera reacted in a fashion indicating that they did not represent any of the HLA-A or -B series and probably not the HLA-C series either. The corresponding antigenic determinants were tentatively designated Ek-1 to Ek-11. The pattern of these antigens among the cell lines is given in a table and it seems apparent that the Ek-series considerably increases the cell identification potential of the typing results. So far, five of the sera determining the Ek-antigens have failed to be blocked by anti-beta-2-microglobulin, indicating that the antigens do not belong to the HLA-A, B or C series. Preparatory work for HLA-D typing is under way.     

Membrane Antigens Characteristic of Human Lymphoid Cells in Established Cultures

Antisera specific for human lymphoid cells in tissue culture were prepared. Rabbits were immunized with a lymphoid cell membrane fraction solubilized by papain and purified by physicochemical means. After appropriate absorption (with peripheral blood leukocytes of the same HL—A type) the antisera reacted with all established lymphoid cell cultures tested, but not with peripheral blood leukocytes.
The antigen(s) reacting with these antisera seem to be unique for cells in culture and appear at the time the cell becomes an established cell line. The antigens were not detectable in normal PBL, non-lymphoid human cell lines in tissue culture or in normal human serum or fetal calf serum, but were present in several leukemic cell samples.     

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.     

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.     

Subpopulations of Cells Involved in the Human Mixed Lymphocyte Culture Response

Various subpopulations of human peripheral lymphoid cells were tested for their role in the mixed lymphocyte culture (MLC) response. Preparations of lymphocytes enriched for T cells, separated by a rosette sedimentation technique, were usually found to exhibit higher MLC responses than preparations enriched for B cells. The relatively high responses of B-cell-enriched preparations could largely be explained by contaminating T cells (approximately 5%) whose MLC reactivities were strongly enhanced by autologous non-T lymphocytes. Cell preparations enriched for B cells were found to be more potent stimulations of MLC responses than T-cell-enriched preparations in most responder-stimulator cell combinations. An MLC response could only be elicited in the presence of cells with characteristics of monocytes/macrophages. These cells could be derived from either the responder or the stimulator.     

Antigen Presenting Cells in Human Decidual Tissue: III. Role of Accessory Cells in the Activation of Suppressor Cells

ABSTRACT: Human decidual antigen presenting cells (DAPCs) exposed to fetal cells in vitro induce generation of suppressor T cells among a population of peripheral blood lymphocytes. Human lymphocyte antigen (HLA)-class II positive antigen presenting cells were isolated from early normal human decidual tissue and from peripheral blood (PAPCs) by adhering Ficoll-Pa-que separated cell suspensions to fibronectin. In contrast to PAPCs, DAPCs pulsed with fetal antigens induced a radio-sensitive, Leu 1,2-positive T suppressor cell population. A nylon wool adherent B cell population is required during the in vitro induction of the suppressor cells. These suppressor cells impair primary mitogen and mixed lymphocyte culture (MLC) responses, generation of anti-trinitrophe-nyl (TNP) cytotoxic T lymphocytes, and antibody response of autologous and allogeneic lymphocytes. Only intact viable embryonic cells can effectively confer upon DAPCs the ability to induce T suppressor cells. The T suppressor cell induction by DAPCs primed with fetal antigens is restricted by the major histocompatibility complex. Our results show that the HLA-DR molecules are the most prominent restriction elements.     

Recognition of the Species of Origin of Cells in Culture by Mixed Agglutination: III. Identification of the Cells of Different Primates

Cultured tissue cells from different primates may be identified by means of the mixed agglutination reaction using suitable antisera prepared in rabbits and in Old World monkeys. In this way it has been possible to differentiate human, Rhesus, Patas and Vervet monkey cells. Several cell lines thought to be derived from Rhesus monkey tissue have been shown to have human species antigens, but not Rhesus monkey antigens, suggesting that the original Rhesus monkey cells have been replaced by human cells. Antigens similar to the human A and B blood group antigens have been identified on Rhesus monkey tissue cells ex vivo, and these have been shown to persist in long-term cultures of Rhesus monkey cells.     

Human Helper T Cell Lines Established by Coculture of Normal Human Cord Leukocytes with an HTLV-Il-infected Rabbit Leukocyte Cell Line (Ra-IIA)

Three helper T cell lines, designated CR -IIA (CR-IIA-1, CR-IIA- 2, and CR-IIA-3), were established by coculturing nor mal human cord leukocytes with a lethally irradiated HTLV II (human T lymphotropic virus type II)- infected rabbit leukocyte cell line (Ra-IIA). CR IIA had a normal human karyotype and expressed the surface markers CD3(+), CD4(+), CD8(-), CD19(-), CD25(+) and HLA- DR(+), confirming their helper T cell nature. CR- IIA cells were all free of Epstein- Barr virus nuclear antigen and were im-muno reactive with serum samples from HTLV- I- or HTLV-Il- infected patients and with anti HTLV- I, p19 or p24 anti body. The provirus genome of HTLV-II was detected in these cell lines by the polymerase chain reaction combined with a digoxigenin- enzyme- linked immunosorbent assay. Electron microscopy of CR-IIA-1 cells revealed a few im mature type C virus particles. These results suggest that HTLV- II was transmitted from the infected rabbit leukocyte cell line to human cord helper T lymphocytes with the development of immortalized HTLV - II- producing T cell lines. Acta Pathol Jpn 42: 347–352, 1992.     

Human Thymic Epithelial Cells in Serum-Free Culture: Nature and Effects on Thymocyte Cell Lines

Thymic epithelial cells (TEC) have been cultured for several months and/or for 4 to 5 transfers in a growth factor-defined serum-free medium without concurrent growth of other cell types. The use of monoclonal antibodies and αMAM-6 indicated that the majority of TEC were of medullary origin. The vast majority of cells were positive for LFA-3 and class I, and class II expression, was low or absent. Supernatants from the cultures were shown to contain IL-1ß, IL-6, and M-CSF. Coculture of cloned subpopulations of thymocytes and TEC showed effects of TEC and of secreted ILs on thymocyte proliferation. High percentages of TEC were able to bind DN, DP, or SP thymocyte populations, partly via CD2-LFA-3 adhesion. Thus, it is possible to culture TEC without unknown serum factors and with maintenance of functional activities.     

Mixed Lymphocyte Culture Search for HLA-D Homozygous Typing Cells in the Hungarian Inbred Population of Ivád

Six functionally HLA-D homozygous typing cells were identified by a restricted investigation into the Hungarian inbred population of Ivád. These putative HLA-D homozygous typing cells were then tested against a highly selected Scandinavian population sample of 60 individuals previously typed by histocompatibility reference reagents. The different HLA-D specificities could thus be identified: one closely matching HLA-Dw5, another resembling the Oslo LDoH specificity, while the last seems to be unique. Only one of the typing cells thus ascertained were HLA-B homozygous and were selected on the basis of the Ivád family structure and not on the basis of serological HLA typing.     

干扰素和肿瘤坏死因子对人脐静脉内皮细胞表达HLA-Ⅱ类抗原的影响

采用cell-ELISA法对IFN-α、γ及rTNFα单独或协同影响人脐静脉内皮细胞（HUVEC）表达HLA-Ⅱ类抗原作了定量观察。结果表明,IFNγ直接刺激 HUVEC可依浓度和时间依赖的方式提高其 HLA-Ⅱ类抗原表达量,而rTNFα、IFNα单独处理HUVEC无效。当rTNFα和IFNγ同时诱导时,对HUVEC表达HLA-DR、DP、DQ均表现抑制作用。但是,当先用IFNγ诱导HUVEC 24h后,再加入rTNFα则发现DR、DP、DQ的表达升高,起促进作用。     

Location of Feline Leukemia-Sarcoma Group-Specific Antigen in Infected Human Tissue Culture Cells

Feline leukemia-sarcoma group-specific antigens were located in human embryo cells infected with feline leukemia and feline sarcoma viruses. This was done by using the fluorescent-antibody and enzyme-labeled antibody techniques at both light and electron microscopic levels. The antigens were found to be exclusively intracytoplasmic, diffuse, and located in discrete punctate foci.     

Characterization by Monoclonal Antibodies of the Cytotoxic Effector Cells in Human Peripheral Blood Mononuclear Cells Reactive against Anchorage-Dependent Tumour Cell Lines

The effector cells for spontaneous cytotoxicity against anchorage-dependent human or mouse tumour cell lines in a 72-h iododeoxyuridine-release assay by normal human peripheral blood cells (PBMNC) or monocyte-enriched fractions were analysed by the use of monoclonal antibodies. PBMNC or adherent or elutriated monocyte-enriched populations of PBMNC were depleted of monoclonal antibody-reactive cells by complement-dependent lysis or separated into monoclonal-antibody-positive or -negative subsets by an indirect rosetting technique followed by Ficoll-Hypaque density gradient separation. The experimental data indicated that in both PBMNC and monocyte-enriched populations, an appreciable proportion of the effector cells with cytolytic activity against adherent human or mouse tumour target cells were positive with B73.1.1 (an antibody with a high degree of selectivity for natural killer (NK) cells), B43.4.1 (or OKM1), and with OKT11a (an antibody recognizing the receptors for sheep erythrocytes), and had the morphology of large granular cells, which have previously been shown to mediate NK activity. These effector cells were mostly negative for BRL.1, BRL.2, B52.1.1, B44.1.1, B13.4.1 and DR antigens, unlike classical monocytes. Some cells which are cytotoxic for the adherent mouse, SV-40-transformed kidney tumour line, TU-5, may bear B52.1.1 or other monocyte-like antigens. Taken together, these results indicate that, in monocyte-enriched populations, both NK cells and monocytes have cytotoxic effector activity against various human and mouse adherent target cell lines.     

正常人外伤脾多向混合淋巴细胞培养产生较高效价γ干扰素

三例正常人外伤猝死者,取其全脾细胞加无血清培养基作多向混合淋巴细胞培养,不加任何干扰素诱生剂,37℃5％CO_2中培育72h后,产生出γ干扰素(1FN-γ)的效价达10240IU/ml,比用一般诱生剂的产量高。这种多向混合淋巴细胞反应的转化率>48％。文中讨论了MLC产生IFN-Y的机理,多向MLC产生较高效价IFN-γ的原因以及与所谓混合淋巴细胸肿瘤细胞培养(MLTC)产生IFN的类型和过程的不同特点。     

Spontaneous Cytotoxicity by Monocyte-Enriched Subpopulations of Human Peripheral Blood Mononuclear Cells against Human or Mouse Anchorage-Dependent Tumour Cell Lines

Human peripheral blood mononuclear cells (PBMNC) were found to be cytotoxic for mouse or human anchorage-dependent target cell lines in a 48-72 h [¹²⁵I)iododeoxyuridine (IUDR) release assay. Unfractionated, adherent or nonadherent cells had significant levels of cytotoxicity, as did cells fractionated according to size into 'lymphocytes' or 'monocytes' by elutriation. Intermediate size cells, not enriched for monocytes, had high levels of cytotoxicity. In all fractions tested, including adherent populations, some cells with the morphology of large granular cells were observed. Treatment of all fractions with interferon (IFLrA, a purified. recombinant α-IFN) boosted cytotoxicity against four target cells lines. Treatment with lymphokines containing putative'macrophage-activating factor'(MAF) also enhanced cytotoxicity in fractions depleted of monocytes. Culture in fetal bovine serum enhanced cytotoxicity mainly in unfractionated and nonadherent PBMNC. These experiments indicated that N K-like cells can be appreciable contaminants in clutriator-purified monocyte-enriched or adherent cell populations and thereby contribute to observed cytotoxicity, particularly after pretreatment with IFN or other stimulatory factors.     

Adherence of Burkholderia pseudomallei Cells to Cultured Human Epithelial Cell Lines Is Regulated by Growth Temperature

We have investigated the adherence of Burkholderia pseudomallei, cultured under a number of different conditions, to six human epithelial cell lines. While several complex medium compositions had relatively little effect on adherence, growth at 30 degrees C was found to significantly increase adherence to all cell lines relative to that of cultures grown at 37 degrees C (P < 0.001).     

Human Leukocyte Antigens (HLA) Class I and Class II on Sperm Cells Studied at the Serological,Cellular, and Genomic Levels

ABSTRACT: The expression of human leukocyte antigens (HLA) on highly purified human ejaculated sperm cells was studied using the sensitive enzyme-linked immunosorbent assay (ELISA) technique and a wide panel of monoclonal antibodies to class I and class II HLA. In addition, the stimulatory capacity of these cells was tested in mixed cultures of lymphocytes and spermatozoa, and the levels of RNA homologous to the HLA class I and class II genes were determined. The results obtained using the ELISA indicate that the class I and class II HLA serologically defined antigens are weakly expressed on the cell surface of the mature spermatozoa. Highly purified sperm cells consistently stimulated heterologous lymphocytes but not when HLA-DR compatibility was observed between stimulator and responder. The proliferative response of lymphocytes induced by sperm cells was lower than the response obtained in a lymphocyte-lymphocyte combination, though the kinetics of the response were similar in both cases. In addition, it was found that spermatozoa contained RNA species homologous to HLA class II DRβ and DQβ genes sequences but not to HLA class I sequences. The levels of these RNA species were significantly reduced after interferon stimulation. Lymphocytes that served as positive control were found to contain RNA complementary to both HLA class I and class II genes.     

Antigenic Specificities Acquired from the Growth Medium by Cells in Tissue Culture

Studies employing quantitative complement fixation have shown that HeLa and other mammalian cells grown in tissue culture adsorb serum protein components from the growth medium. The serum proteins are not completely removed by vigorous washing. Upon injection of extensively washed cells into rabbits the bound serum proteins give rise to specific antibodies detectable by gel diffusion and complement fixation. With horse serum in the growth medium one of the cell-bound components was identified as horse γ globulin. Evidence was obtained that specific antibodies to bound serum antigens can contribute to complement-dependent kill of the cells in vitro. These observations suggest one possible mechanism for the acquisition of antigenic relationships by diverse cell lines grown in tissue culture.