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 
3
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
co-culture 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.