Fluorescence staining of freshly dispersed cells will also be 
evaluated. The presence of plasmid DNA will be confirmed by PCR 
of DNA from tumor tissue, peripheral blood lymphocytes, or in 
autopsy specimen tissue. If sufficient tissue is available, RNA 
will be isolated and examined for the presence of HLA-B7 mRNA by 
PCR or SI nuclease analysis. 
11.3 Analysis of immune response. 
Cytolytic T cell activity will be evaluated by incubation of 
peripheral blood lymphocytes with 51 Cr labeled HLA-B7 transduced 
EBV lines or malignant melanoma cells from the patient. The 
presence of antibody will be evaluated by FACS analysis of a 
matched pair of HLA-B7 + or HLA-B7 - cell lines. In some 
instances, lymphocytes will be isolated directly from the tumor, 
expanded in tissue culture, and analyzed for cytolytic function. 
Tumor biopsies at 7-14 days after treatment will be analyzed by 
immunohistochemistry . If possible, we will attempt to expand 
draining lymph node T cells or TIL cells to test their cytologic 
function. When possible, we will derive autologous cell lines to 
be used as targets in a 51 Cr release assay. Finally, every 
attempt will be made to excise tumor tissue prior to treatment 
for diagnosis, immunohistochemistry, and cryopreservation and to 
evaluate delayed type hypersensitivity reactions to the tumor 
before and after treatment. 
Recombinant DNA Research, Volume 15 
[423] 
