M. A. V. Crimmins

Human Cytolytic T-Lymphocyte Clones and Their Function-Associated Cell Surface Molecules

Cytolytic T lymphocytes (CTLs) are important effectors in the recognition of viruses,1 allografts,2 and some tumors.3 A molecular understanding of the CTL-target cell interaction therefore may be relevant to the etiology and/or treatment of a variety of disease states. Tissue culture techniques, first reported by Gillis and Smith,4 have allowed the generation of long-term T-cell lines that retain function. A number of human long-term cytolytic T lymphocyte lines have been generated by continued stimulation of peripheral blood lymphocyte lines with “foreign” cells in the presence of the T-cell growth factor interleukin 2 (IL-2).5,6 We have used CTL lines and clones to define target antigens recognized by human allogeneic lymphocytes, to correlate lymphocyte phenotype with antigen specificity, and to generate monoclonal antibodies that block lymphocyte function. Our findings have provided new insights into the cell surface molecules involved in the CTL-target cell interaction. In this chapter we describe our methodologies for the generation and maintenance of CTL lines and clones and the use of the cells in concert with monoclonal antibodies to define and analyze function-associated cell surface molecules.

The role of CD18 in phorbol ester-induced human monocyte-mediated cytotoxicity

Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.

Molecular Characterization of Allospecific Cytotoxic T-Lymphocyte Recognition Sites in the HLA-A2 Molecule Using Oligonucleotide-Generated Site-Specific Mutants

The complexity and fine specificity of the allospecific T-cell response generated against the human HLA-A2.1 molecule and the characterization of the antigenic determinants that anti-HLA-A2 alloreactive CTLs recognize in the molecule has been analyzed using as targets, cell lines expressing HLA-A2 CTL-variants and the human rhabdomyosarcoma cell line (RD) transfected and expressing HLA-A2 mutants obtained by site-directed mutagenesis.