Figure 4. Analysis of immunologic function of primary T cells 
after transduction with Rev M10 or ARev M10 vectors. T cells 
were stimulated with immobilized anti-CD3 and supernatants 
harvested after 10 and 24 hours of culture. (A) Supernatants 
were analyzed for TNFa activity using the L929 bioassay. (B) 
Cytolytic function of transduced T cells were evaluated using 
... . Cl 
anti-CD3 crosslinking of T cells to Fc receptor positive Cr- 
labeled P815 tumor targets . 
III. Expression of recombinant genes in T cells following 
biolistic injection 
Although retroviral vectors are well-established as a means 
to introduce genes into T cells, successful transduction requires 
the activation of T cells which may accelerate their subsequent 
cell death. Because of this problem, we have begun to develop 
techniques to introduce DNA into primary human peripheral blood 
lymphocytes using electroporation. To accomplish this, 
unstimulated or activated cells were transduced with biolistic 
injection under conditions optimized for gene expression. The 
details of this protocol are included in Appendix IV, Section 8. 
When these experiments are performed, recombinant gene expression 
can be detected in 1-10% of human peripheral blood CD4 + T 
lymphocytes. From this population, 0.1% of cells can be cloned by 
limiting dilution which show G418 resistance with the RSV-tar 
plasmid proposed for use in these studies. Experiments are on- 
going to determine whether these cells are resistant to HIV 
Recombinant DNA Research, Volume 18 
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