CELL-MEDIATED DELIVERY OF PROGRAMMED CELL DEATH SIGNALS FOLLOWING GENE TRANSFER OF A MEMBRANE-ANCHORED SINGLE-CHAIN ANTIBODY SPECIFIC FOR CLASS I MHC

Abstract

821 Introduction. Gene transfer of death signaling molecules has not been reliably successful in protecting allogenic cellular and tissue transplants from immune rejection. We describe the use of a membrane anchored, single-chain antibody (5H7Fv-GPI) that delivers human lymphoid programmed cell death (PCD) signals by ligation of class I MHC. 5H7Fv-GPI is derived from 5H7, a mAb specific for the α3 domain of class I MHC that induces veto-like PCD signals in human lymphoid cells. Derivation of a single-chain version of 5H7 provides a means for achieving cell surface expression of the 5H7 idiotype on cells that lack the machinery to properly assemble whole mAb. Methods. Chinese Hamster Ovary (CHO) cells were stably transfected with pC5H7Fv-GPI (5H7Fv coupled to CD58-derived glycosylphosphotidylinostitol (GPI)). Lymphoid PCD was assessed by co-culture of CHO-v5 (pC5H7Fv-GPI transfected) or CHO-neo (control vector pCDNA 3.1 transfected) cells with lymphoid targets. Competitive release of target cells from CHO cells was achieved by addition of A2.4, an anti-idiotypic mAb specific for the 5H7 idiotype. PCD morphology was determined by fluorescence microscopy following ethidium bromide/acridine orange staining and confirmed by Annexin V-FITC flow cytometric measurements. Results. CHO-v5 cells actively bound recombinant-derived soluble class I MHC, as determined flow cytometry. CHO-v5 cells induced PCD in Jurkat and BeVD lymphoid tumor cell lines, but not in control Daudi cells (class I MHC deficient). Jurkat cell PCD is not observed with use of whole 5H7 mAb, suggesting that 5H7Fv-GPI provides a modified or augmented PCD signal, when compared to parental mAb. Observations from several studies indicate that 5H7Fv-GPI delivers PCD signal with a potency comparable to that of the parental 5H7 mAb. Human PBMC were also susceptible to class I MHC-induced PCD following exposure to CHO-v5 cells. Preactivation of PBMC with immobilized anti-CD28 and anti-CD3 mAbs augmented PCD after co-culture with CHO-v5, a finding similar to that observed with the 5H7 mAb. The specificity of class I MHC signalling was addressed by the addition of excess A2.4 mAb, which reduced lymphoid PCD to background levels. Splenocytes derived from B6 mice transgenic for class I HLA-B27 also underwent PCD after coculture with CHO-v5, while splenocytes from transgene negative littermates did not show PCD. Thus, the inability of CHO-v5 to induce PCD in Daudi cells, B6 non-transgenic mice splenocytes, and the inhibition of PCD by A2.4 mAb confirm that CHO-v5 mediates PCD via class I MHC. Conclusions. 1) Cell surface expression of the 5H7Fv-GPI gene reconstitutes the 5H7 idiotype and retains ability to bind to class I MHC. 2) 5H7Fv-GPI PCD induces PCD in lymphoid cells that express class I MHC 3) Gene transfer of 5H7Fv-GPI provides a promising approach for modulating immune responses to human allografts.