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 
Direct gene transfer and expression of the HLA-B7 gene may 
sensitize the patient to HLA-B7 and lead to the generation of an 
immune response to this antigen. Limiting dilution analysis 
(LDA) will be utilized to evaluate alterations in the frequency 
of helper and cytolytic T cells for HLA-B7 in the peripheral 
blood following direct gene transfer. Peripheral blood 
lymphocytes will be isolated and cryopreserved prior to, and at 
4-week intervals, following the initial direct gene transfer. At 
the completion of treatment, samples of PBL from each time point 
will be simultaneously evaluated for responsiveness to HLA-B7 by 
culturing PBL, under LDA conditions, with autologous EBV-B cells 
transduced with the HLA-B7 gene. Antigen specific elaboration of 
IL-2 or generation of CTL to HLA-B7 positive target cells will be 
the indices evaluated in these studies. 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 
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Recombinant DNA Research, Volume 17 
