I 
f. Absence of autonomous growth - 
IL-2 will be withdrawn from an aliquot of TIL for one week to make certain that 
they do not exhibit autonomous growth 
I; 
g. Cytotoxicity - 
targets include K562, Daudi, allogeneic and autologous tumor 
h. Phenotype - 
Flow cytometric analysis will be performed using the following panel of 
antibodies: CD3/CD8, CD3/CD4, CD3/CD56, CD16/CD56, CD8/CD38, CD8, 
Leu8, CD8/CD25, CD8/CD116, CD8/anti HLA-DR, CD8/CD4SRA, CD8/ 
CD29, CD3/TCR. 
i. mRNA expression- 
whole cellular RNA will be extracted from TIL and PBL using the guanidinium - 
CsCl method and 20/xg separated on agarose gels. After transfer to nylon 
membranes, these will be probed with 32 P-cDNA for TNFo;, IL-16, IL-6, 
IL-4,IL-2 and IL-7. 
j. cytokine - 
An aliquot of TIL(5x 107ml) will be cultured in 6 well Costar plates in AIM V 
+ 1000 u/ml IL-2 for 48 hr and the cell-free supernatants assayed for TNFa, IL- 
16, IL-6, IL-4, and IL-7 using ELISA assays. 
k. HLA typing- 
Prior to patient infusion, TIL and PBL will be HLA-typed with freshly retrieved 
patient PBL as a safeguard against laboratory labelling error. 
15.6 CLINICAL STUDIES 
In addition to evaluating clinical response (see section 13), the following studies 
will be performed. 
(a) BLOOD - 10 cc peripheral blood will be obtained 1,4,7,14,30,60,90 and 120 
days after TIL infusion. PBL will be isolated by Ficoll-Hypaque centrifugation 
and DNA prepared. Semiquantitative PCR analysis will be performed using 
appropriate primers. The number of neoR gene copies of LNL6 and GIN will 
be determined. 
(b) TUMOR AND ADJACENT NORMAL TISSUE - Patients with easily 
accessible, subcutaneous melanoma or renal cell carcinoma metastases will be 
consented to undergo open or needle biopsy of the tumor and adjacent muscle or 
uninvolved skin. The number of samples will depend on available 
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