William Boyle

Isolation of macrophages from human placenta

Human placentae have been extracted with combinations of enzymes to optimize the release of mononuclear phagocytes. A mixture of trypsin-DNAase used in sequential extraction was found to provide the best yield of adherent cells which were stable in culture. The majority of adherent cells exhibited phagocytic function and expression of receptor for IgG-Fc (FcR). Subsequent studies established that these functions were co-expressed by the same cells. The FcR+ cells were also shown by immunofluorescence with monoclonal antibodies to display monocyte-macrophage distinctive antigens and class I and class II MHC antigens. The placenta has thus been shown to provide a rich source of class II-positive macrophages suitable for immunological studies.

Class II MHC antigen-positive macrophages from human placentae suppress strong MLR and CML reactions

Placental adherent cells (PAC), derived by mild enzymic digestion of human placentae and adherence to plastic, were tested for their ability to suppress MLR and CML reactions. Stimulation of allogeneic T cells by cord blood mononuclear cells or by a strongly stimulatory B lymphoblastoid cell line were consistently inhibited by all PAC preparations tested. PAC were fractionated by Fc rosetting and Percoll gradients to produce a number of bands. Suppression correlated with the content of macrophages in each band and was strongest in band 5 which generally contained 94-100% macrophages. The suppressive effect was not inhibited by indomethacin. The possible role of macrophages in suppressing immune reactivity in the placenta is discussed.

A micro-version of the 51Cr release assay for cytotoxic lymphocytes.

A micro-version of the 51Cr release assay for cytotoxic lymphocytes has been devised, which is conducted in Terasaki plates, in a total reaction volume of 8 microliter. The conditions necessary to make the microassay equivalent in sensitivity and reproducibility to the standard macroassay are described. In comparison with the standard macroassay, the microassay provides a 10-fold reduction in the number of effector cells employed and represents a 50-fold reduction in reaction volume. The assay has been particularly valuable in the saving of antisera when these are employed to block target cell lysis, or to selectively inactivate cells of a particular phenotype in the effector cell population.

Production and purification of human indoleamine 2,3-dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO

The human indoleamine 2,3‐dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa‐histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme‐containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti‐HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN‐γ‐stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells: I. Metabolic inactivation impairs both CD and LD antigen signals

Abstract The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T c ) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T c responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T c precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T c from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T c . It is concluded that, in addition to the traditional view that these treatments damage the “LD” signal to helper T lymphocytes, metabolic inactivation also impairs recognition of “CD” determinants by precursor T c .

Cytolytic t lymphocyte responses to metabolically inactivated stimulator cells. II. Effect of a soluble "costimulator" factor(s) in primary and secondary mixed leukocyte culture.

Abstract The ability of a concanavalin A-stimulated spleen cell supernatant (“costimulator”) to overcome the effects of impaired CD and LD antigen presentation by metabolically inactivated stimulator spleen cells was examined in the primary and secondary cytolytic T lymphocyte (Tc) response. (i) Cells inactivated by ultraviolet irradiation or mild glutaraldehyde treatment, which were unable to stimulate primary cytolytic activity on their own, generated near maximal responses in the presence of costimulator. The 30-fold lower efficiency of splenic membrane fragments as antigen in primary MLC with the supernatant indicated that the damage to immunogenicity caused by membrane isolation was not equivalent to that caused by uv light and glutaraldehyde, as has previously been assumed. (ii) Comparison of the relative effects of antigen and costimulator demonstrated that costimulator played the dominant regulatory role in primary MLC, increasing sensitivity to suboptimal antigen doses 10- to 30-fold; neither antigen nor the supernatant appeared preferentially to control the strength of the secondary response. (iii) Metabolically inactivated adult and untreated neonatal spleen cells failed to release costimulator activity in response to concanavalin A. However, the ability of the neonatal cells to induce a primary cytolytic response suggested that costimulator production by the stimulator cells themselves is not essential for primary Tc activation, and supports the hypothesis that the lack of primary immunogenicity of inactivated spleen cells reflects their failure to induce costimulator production by the responder population.