translated to produce carboxyl terminal truncation products that will be used in 
immunoprecipitation studies. In addition, two monoclonal antibodies (4D4 and 9H9) 
recognizing different portions of the LDL receptor have been obtained and ascites produced to 
serve as positive controls for these assays. 
It is possible, albeit unlikely, that the antibody response to the transplanted cells will 
be sufficiently intense to cause a systemic reaction or serum-sickness type reaction. This 
would be initially evaluated through the detection of anti-LDL receptor antibodies, however, to 
confirm that the transplant was responsible for the reaction, further studies would be 
performed. Circulating immune complexes would be isolated from serum through differential 
polyethyleneglycol precipitation. These complexes can then be probed for specific antigens 
(LDL receptor) related to the transplant using Western blot analysis and anti-LDL receptor 
monoclonal antibodies. 
Cellular Studies. Transplant recipients will be analyzed for the development of T cell 
immune responses to the transplanted cells. 
The requirements for an antigen to use in studies identifying T cell immune responses to 
the LDL receptor are very different from those needed for serologic studies. Glycosylation and 
conformation are not important, however, removal of substances capable of causing non- 
specific lymphocyte activation (such as endotoxin) is essential. One method is to employ 
autologous cells for antigen presentation and as targets of cellular cytotoxicity. However, it 
would be technically easier to have isolated fragments of the receptor to use as antigen-specific 
stimulants in PBMC cultures. With the recent cloning of LDL receptor fragments into the 
bacterial expression vector, pMALc Rl, and the ability of this unique fusion protein produced by 
it, receptor fragments can be isolated from contaminating bacterial proteins and 
lipopolysaccarides can be used in lymphocyte proliferation studies. 
LDL receptor stimulations of PBMC will be performed using several concentrations of 
the recombinant proteins produced in the pMALc Rl expression system. T cell responses will be 
determined by measuring Thymidine incorporation in the PBMC. Specificity of the immune 
response for mutational areas of the LDL receptor will be determined by comparing T cell 
responses to whole native versus normal LDL receptor. In addition, synthetic peptides 
corresponding to the regions of the mutations in the LDL receptor will be used to stimulate the 
PBMC to determine if the T cell responses to receptor are specific for these areas. Cytotoxic 
cellular immune responses will be determined by transfecting autologous cell lines 
(lymphoblastoid or fibroblasts) from the subject with the LDL receptor. These cells would then 
be 51 Cr labeled and used as targets in a chromium release assay. 
Through these methods it is hoped that any form of immune response to the transplanted 
hepatocytes would be identified and characterized during the course of the experiment and the 
results of such an immune response could be correlated with outcome parameters to determine 
if it effected the transplanted cells. It may also be important to correlate observed immune 
responses to the LDL receptor defect phenotype of the recipient. This would clarify whether 
receptor negative individuals with gene deletions or nonsense mutations are more likely to 
develop immune reactions to transplanted proteins than recipients who have defective, but 
immunologically intact endogenous proteins. 
Recombinant DNA Research, Volume 15 
[191] 
