PURIFIED TRUNCATED RECOMBINANT HLA-B7 MOLECULES ABROGATE CELL FUNCTION IN ALLOREACTIVE CYTOTOXIC T LYMPHOCYTES BY APOPTOSIS INDUCTION1,2

Abstract

Soluble MHC class I molecules are ubiquitous in human body fluids, including serum, urine, sweat, and cerebrospinal fluid. However, their biological function has remained unresolved. Membrane-derived human soluble MHC molecules (soluble human leukocyte antigen; sHLA) have been shown to induce apoptosis in alloreactive cytotoxic T lymphocytes (CTL). Here we report the efficacy of recombinant soluble HLA-B7 (rsHLA-B7) to modulate T-cell function. Primers of HLA-B7 were designed to allow amplification of a cDNA lacking the transmembrane and cytoplasmic domains yielding a truncated gene. rsHLA-B7 molecules were expressed in the human myeloma cell line 721.221 and purified by affinity chromatography using the BB7.7 mouse monoclonal antibody. CTL were generated from peripheral blood lymphocytes derived from healthy blood donors by stimulation with irradiated Epstein Barr virus-transformed HLA-B7-positive B cells. CTL were preincubated with rsHLA-B7, and cytotoxicity and apoptosis were tested according to standard procedure. A total of 2 x 10(6) cells/ml secreted 10 microg/ml rsHLA-B7 as determined by a conformation-dependent ELISA, suggesting that rsHLA-B7 do not require the transmembrane and cytoplasmic regions for proper folding. After purification by affinity chromatography, rsHLA-B7 induced apoptosis in anti-HLA-B7 CTL, but not in anti-HLA-A2-specific, CTL. As a consequence, allorecognition of target cells by the CTL was significantly blocked. Recombinant sHLA are sufficient binding cues for T cells, which efficiently induce apoptosis and block allorecognition of target cells by CTL. Thus, recombinant sHLA molecules may become a valuable new modality for specific immunological therapeutic intervention.