dose is an important parameter in the magnitude of the immune response, such 
that at higher Dose Levels, the contribution of lxlO 6 irradiated tumor cells 
used in DTH testing may be insignificant to the immunizing dose of 10 8 or 10 9 
cells . 
7.2.C. DRAINING LYMPH NODE ANALYSIS - A consistent feature of 
our preclinical model is substantial enlargement of the draining lymph nodes 
of the vaccibe site by day seven after vaccination with GM-CSF transduced 
tumor vaccine. (Appendix 14. A. 4) The amount of enlargement is substantially 
greater than when irradiated non-transduced tumor is used alone. Histologic 
analysis shows both paracortical hypertrophy (T cell dependent zone) as well 
as some increase in the germinal center regions (B cell areas). Functional 
studies demonstrate a profound increase in the proliferation of CD4 + T cells 
relative to that of unprimed lymph node cells, or those primed with non- 
transduced irradiated tumor alone. Analysis of the draining lymph node cells 
for endogenous cytokine mRNA suggests the initiation of a cascade of cytokine 
production including IL4 and IL6. Finally, by day 7, tumor specific CD8 + CTL 
may be derived after in vitro expansion from draining lymph node cells. These 
observations delineate the early events that characterize the initial 
"priming" events after tumor cell/GM-CSF vaccination in murine models. This 
constellation of parameters will be examined in this study as an indication of 
immunological activation, as well as to potentially derive reagents such as 
cytotoxic T-cell clones that may aid in defining the tumor specific peptides 
that are the target of immunologic recognition. For these reasons, if the 
right draining inguinal nodes increase two fold or greater during the first 
treatment cycle lymph node biopsy will be performed in this study and assayed 
as described in the next section. 
Recombinant DNA Research, Volume 17 
[ 327 ] 
