Stephen T. Licence

Analysis of monoclonal antibodies that recognize γδ T/null cells

Abstract Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γ δ TcR established the majority of null cells are γ δ T cells. Use of this mAb in further comparisons demonstrated the γ δ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2 − γ δ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.

THE BEHAVIOUR OF MONOCLONAL ANTIBODIES IN THE FIRST INTERNATIONAL PIG CD WORKSHOP REACTING WITH GAMMA DELTA /NULL T LYMPHOCYTES IN THE BLOOD OF SLAB/B LINE PIGS

Further studies were carried out on the monoclonal antibodies (mAbs) from the First International Swine CD Workshop which react with gamma delta Null T-lymphocytes, defined by the binding of mAb w020/141 (MAC320) as Swine Workshop Cluster number SWC6. Studies were also carried out on several other mAbs from the same workshop which identify other CD antigens, but whose binding is not restricted exclusively to gamma delta Null T-lymphocytes. The first group consists of 11 mAbs (w021, w022, w059-w065, w105 and w117) and the second group of 18 mAbs (w008, w026, w056, w067-w071, w080, w091-w094, w110, w111, w118, w119 and w121). All mAbs were characterised by binding to peripheral blood lymphocytes (PBL) from normal, sham-thymectomized (STx) and thymectomized (Tx) pigs of the Babraham SLAb/b line and by their overlap, using two-colour immunofluorescence with biotinylated mAb MAC320 (w020/141), which identifies all gamma delta Null cell T-lymphocytes and the Null cell subpopulation identified by MAC319 (w021). The Null cell-specific mAbs were also used in inhibition studies of MAC319 and MAC320 binding and by staining PBL with pairs of mAbs together with either MAC319 or MAC320. Based on these data we suggest a putative relationship of the Null cell subsets defined by these mAbs with each other.

Staining of pig lymphocytes subpopulations with acid α-naphthyl acetate esterase

Abstract The use of α-naphthyl acetate esterase (ANAE) as a T cell marker in some other species and the broad correlation of incidence of ANAE-positivity and E rosette-formation in the pig suggest that ANAE-staining may be a T-cell marker in the pig. However, by studying the staining of lymphocytes within a variety of rosettes in fixed preparations a similar incidence of pig blood lymphocytes were found to be ANAE + among T cells (E rosettes formed in dextran), B cells antiglobulin rosettes) and Fc-γ receptor-bearing B and T cells (EA rosettes in saline and dextran): complement (C′) receptor-bearing cells showed a higher incidence of staining than other lymphocytes. Analysis of staining morphology suggested that certain morphologies within the B and T lineages may be confined to subpopulations. Thus ANAE positivity is certainly not a marker identifying blood T lymphocytes but could be of some value indicating subpopulations of B and T lymphocytes.