Possible Use of Established Cell Lines for MLR Locus Typing

MLR histocompatibility typing was performed by in vitro stimulation of lymphocytes from various persons with fresh lymphocytes (irradiated) or with lymphoblostoid cell lines (mito-mycin-treated) derived from donors homozygous at the MLR locus. MLC responses to both types of stimulation correlated strongly suggesting that a) established lymphoblastoid line cells can express products of the MLR loc and b) fresh lymphocytes can be replaced by these long term lines in MLR typing. Two lines (LV-B and MS-B) derived from two MLR homozygous donors (lv and MS) were used throughout these experiments. It is possible that as additional lines are established from such MLR homozygous donors, a panel of typing cells could be created which would serve to standardize MLR typing and allow selection of unrelated but histocompatible donors for transplantation.     

Release of Thy-1 from Human and Murine T-Cell Lines by a Specific Phospholipase

Proteins on the outer surface of cultured human and murine lymphoblastoid T cells were labelled with ¹²⁵I. The labelled cells were incubated with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC). Proteins cleaved from the cell membrane by the enzyme were immunoprecipitated with anti-Thy-1 antibodies, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and identified by autoradiography. A doublet of Thy-1 bands of approximately 16,000 daltons were detected. The result suggests that: (a) Thy-1 is present on the human and murine T cells which we tested, and (b) Thy-1 is attached to the cell membrane via a phosphatidylinositol domain.     

Release of Thy-1 from Human and Murine T-Cell Lines by a Specific Phospholipase

Proteins on the outer surface of cultured human and murine lymphoblastoid T cells were labelled with ¹²⁵I. The labelled cells were incubated with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC). Proteins cleaved from the cell membrane by the enzyme were immunoprecipitated with anti-Thy-1 antibodies, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and identified by autoradiography. A doublet of Thy-1 bands of approximately 16,000 daltons were detected. The result suggests that: (a) Thy-1 is present on the human and murine T cells which we tested, and (b) Thy-1 is attached to the cell membrane via a phosphatidylinositol domain.     

Production of Interferon-β by Murine T-Cell Lines Induced by 10-Carboxymethyl-9-Acridanone

Besides the established T-cell property of producing gamma interferon (IFN-gamma), murine T cells additionally possess the ability to produce IFN-alpha and IFN-beta when appropriate inducers such as 10-carboxymethyl-9-acridanone (CMA) or Newcastle disease virus (NDV) are used. Interleukin 2 (IL-2)-dependent murine T-cell lines, but not purified resting splenic T cells, responded to CMA and NDV with production of IFN-alpha, beta. The IFN production by these T cells was not restricted to a special subset, since T cells expressing the Lyt 1+2- and the Lyt 1-2+ phenotype responded to these inducers with IFN production. After prolonged passaging of the T-cell lines in IL-2-containing medium, the ability to respond to CMA with production of antiviral activity was sustained longer than the ability for concanavalin A-induced IFN-gamma production. Whereas the NDV-induced T-cell supernates contained both IFN-alpha and IFN-beta, the induction with CMA resulted exclusively in the synthesis of IFN-beta by the T-cell lines.     

A Model System for the Analysis of B-Cell Activation and Effector T-Cell Functions

We have attempted to develop an in vitro system where polyclonal B lymphocyte responses could be induced in 'antigen-like' conditions, that is, where surface immunoglobulin-dependent binding mediates interaction with a mitogen. Monoclonal anti-mu and anti-delta antibodies were covalently bound to lipopolysaccharide (LPS) and these complexes were shown to display mitogenic activity. Polyclonal plaque-forming cell (PFC) responses, however, were diminished in cultures stimulated by anti-mu-LPS (but not by anti-delta-LPS) indicating that 'anti-mu inhibition' of terminal B-cell differentiation also applies to 'specific' antibody responses. Moreover, the analysis of the functional activity of monoclonal antibodies to major histocompatibility complex (MHC) class II molecules revealed a surprising synergy between low, non-stimulatory concentrations of anti-mu-LPS (but not anti-delta-LPS) with anti-I-A antibodies. These responses are T-cell dependent and synergy with anti-mu-LPS conjugates can also be obtained with 'naturally' activated CD4+ cells isolated from normal donors. A model of molecular and cellular interactions was derived, which accounts for the present findings and is applicable in antigen-dependent lymphocyte collaboration.     

Guidelines for Reporting and Using Prediction Tools for Genetic Variation Analysis

Computational prediction methods are widely used for the analysis of human genome sequence variants and their effects on gene/protein function, splice site aberration, pathogenicity, and disease risk. New methods are frequently developed. We believe that guidelines are essential for those writing articles about new prediction methods, as well as for those applying these tools in their research, so that the necessary details are reported. This will enable readers to gain the full picture of technical information, performance, and interpretation of results, and to facilitate comparisons of related methods. Here, we provide instructions on how to describe new methods, report datasets, and assess the performance of predictive tools. We also discuss what details of predictor implementation are essential for authors to understand. Similarly, these guidelines for the use of predictors provide instructions on what needs to be delineated in the text, as well as how researchers can avoid unwarranted conclusions. They are applicable to most prediction methods currently utilized. By applying these guidelines, authors will help reviewers, editors, and readers to more fully comprehend prediction methods and their use.     

Lymphoid Cell Lines as Indicators of Lymphokine Generation

We investigated the migration characteristics of the cells of four human lymphoid lines, normal peripheral blood leucocytes (PBL), and normal mouse splenocytes (MSC). Two lines (QIMR-WIL and Namalwa) were actively migratory, as were the PBL and MSC. Migration was inhibited at low temperatures and reduced by inhibition of glycolysis, oxidative phosphorylation and RNA synthesis. The migration of QIMR-WIL cells, PBL and MSC but not Namalwa was inhibited by lymphokine-containing supernatants from phytohaemagglutinin-pulsed MSC or MSC stimulated by allogenic cells. The inhibitor of migration in the supernatants had properties similar to those of human leucocyte inhibition factor but distinct from those of macrophage inhibition factor. QIMR-WIL and normal human PBL were compared as indicators in an indirect leucocyte migration assay. QIMR-WIL cells were the more sensitive responder cells and are an abundant, stable, and uniform cell population for lymphokine detection.     

Characterization of an Lyt-l+ Cytolytic T-Cell Clone Specific for a Polymorphic Domain of the I-Ak Molecule

We have characterized a cytolytic T-cell clone, isolated from a A.TH anti-A.TL mixed lymphocyte culture, which recognized a private determinant of the I-Ak molecule. This specificity has been confirmed by inhibition of effector-target cell interaction by anti-I-Ak monoclonal antibody (mAb). Comparison of the inhibitory capacity of various mAb and the spatial arrangement of their epitopes (defined in previous studies by antibody-binding competition) indicated that the antigenic site recognized by this cytolytic T-cell clone was topologically related to one of the major polymorphic domains of the Ak molecule. This clone expressed the Thy-1.2+, Lyt-1.+, Lyt-1.2+low and I-As- cell surface phenotype. Testing of several rat mAb, screened for their ability to inhibit H-2K/D-specific cytolysis at the level of the effector cells, revealed that two anti-p94, 180 mAb but not various anti-Lyt-2 mAb inhibited the lytic function of this anti-I-Ak T-cell clone.     

Analysis of Neonatal T Cell and Antigen Presenting Cell Functions

ABSTRACT: Neonates are more susceptible to infection than adults and exhibit more intense or prolonged clinical symptoms. The extent to which deficiencies in T cell or antigen presenting cell (APC) function underlie hyporesponsiveness is incompletely understood. Here, immune function of cord blood mononuclear cells (CBMC) from healthy, full-term neonates was compared with adult PBMC. As widely reported, polyclonally-stimulated T cell proliferation was found to be equivalent, while IFNγ responses were markedly lower amongst neonates. Reasoning that such stimuli may elicit responses qualitatively different from those that would be obtained following MHC-dependent, cognate T cell activation, alloantigen-specific responses were evaluated. Strikingly, neonates exhibited IFNγ, IL-4 and IL-10 production equal to adults in short term primary culture. Both the frequency (Fisher’s p < 0.0004) and intensity (<7.5 vs 36.5 pg/ml; Wilcoxon P = 0.005) of alloantigen stimulated IL-5 responses were elevated among neonates, a finding equally evident using irradiated adult or neonatal cells as stimulators. Finally, the relative capacity of neonatal APC as stimulators of cytokine synthesis was assessed by a novel approach using CBMC as both responders and stimulators in MLR. Irradiated neonatal cells consistently stimulated similar proliferative but substantially lower IFNγ responses than did adult APC, independent of responder origin. The data argue; (i) T cells are largely immunocompetent at birth, (ii) accessory cell function is not fully mature, and (iii) the widely observed hyporesponsiveness to pathogens may be primarily due to immaturity of APC function or costimulator molecule expression.     

HLA Class-II-restricted Mycobacterium lepraereactive T-Cell Clones from Leprosy Patients Established with a Minimal Requirement for Autologous Mononuclear Cells

This report describes an effective method for the cloning of Mycobacterium leprae-reactive T lymphocytes with Epstein-Barr-virus transformed autologous B cells as antigen-presenting cells. The two advantages of this method are that it drastically reduces the number of autologous peripheral blood mononuclear cells (less than 10(7) cells) needed to obtain and propagate these T-cell clones (TLC), and that it enables us to expand individual TLC to large numbers of cells (greater than 10(8)). Thus the major obstacles for the cloning of T lymphocytes--especially important with regard to patients--are bypassed. Thus far, TLC from three leprosy patients have been established. These TLC are HLA class II restricted in their M. leprae-directed response. A marked enhancement in antigen responsiveness was observed after further expansion of several TLC, some of which turned from nonresponder into responder TLC. Four tested TLC display strikingly different antigen recognition patterns when tested against a number of other mycobacterial antigens; one TLC so far recognizes only M. leprae antigens.     

The Enrichment of T-Cell Functions in a Lymphocyte Population by Anti-immunoglobulin Columns

Mouse lymphocytes have been fractionated using purified anti-light chain antibodies adsorbed onto plastic beads. B cells were specifically bound on these columns, as evidenced by the absence of anti-light chain immunofluorescent staining of the effluent cells and a two-fold enrichment of T cells as judged by cytotoxicity and fluorescent staining using specific anti-T-cell sera. The effluent population was still functional and showed a 10–30% enrichment with respect to the killing of allogeneic mastocytoma cells. Although only 71% of spontaneous and 58% of immune rosettes formed by unfractionated lymphocytes with sheep red cells could be inhibited by an AKR anti-CBA θ serum, virtually all the rosettes formed by the anti-light chain effluent cells were inhibited. θ-bearing rosette-forming cells from immunized animals were retained to a greater extent Dn the anti-LC as compared with control columns.     

Morphological and Cytogenetic Characterization and N-myc Oncogene Analysis of a Newly Established Neuroblastoma Cell Line

A permanent cell line established from a xenograft of neuroblastoma which occurred in a 5 year old girl was investigated for its morphological and biological characteristics. The cultured cells were tumorigenic in nude mice. Microscopically, each tumor consisted of small round to polygonal cells with irregular nuclei and prominent nucleoli, corresponding to the features of the primary and xenografted tumor cells. Electron microscopic examination revealed that both the transplanted tumor cells and the cultured cells contained scanty microtubules and dense-core neurosecretory granules. Chromosome analysis of this cell line showed monosomy for chromosomes 1,10,19 and X, and structural rearrangements involving chromosomes 8, 17 and 20, in addition to numerous double minutes. The N- myc oncogene was found to be amplified 40 to 80 fold in the transplanted and cultured tumor cells, as well as in the primary tumor cells. In situ hybridization with a digoxigenin labeled uridine-triphosphate N- myc RNA probe detected abundant mRNA in the tumor cells. This neuroblastoma line may become a valuable in vitro experimental model system for studies aimed at better characterization of neuroblastoma. Acta Pathol Jpn 41: 507 515, 1991.