These studies confirmed that IL-4 secretion by A9 tumor was associated with reduced 
tumorigenicity. However, none of the animals were found to be tumor free. 
We proceeded to examine if A94-3 growth would elicit pre-effector cell sensitization in 
tumor-draining lymph nodes (TDLN). Mice were inoculated with 10 6 A94-3 or A9PLJ1 s.c. 
and 9-10 days later TDLN harvested to assess for pre-effector cell sensitization against 
parental tumor. TDLN were activated by the anti-CD3/IL-2 procedure we have previously 
described (see Section 2.1) and their antitumor reactivity assessed by the adoptive 
immunotherapy of established 3-day parental (A9) pulmonary metastases (Table 3). In these 
series of experiments, 4-5 x 10 7 cells were transferred with the concomitant administration of 
low doses of IL-2 (15,000 u i.p. bid x 4d). We found that LN draining A94-3 tumors mediated 
significant regression of A9 lung tumors compared to groups of mice which were not treated 
with activated cells. There was no antitumor reactivity of activated LN cells derived from 
A9PLJ1 bearing animals at the cell dose used. 
Table 3: Adoptive Immunotherapy of Pulmonary Metastases with LN Cells Draining IL-4 
Transfected B16-BL6 
Source of 
LN Cells 
Cells 
Transferred 
Mean # Metastases (SE) 
Exd 1 Exd 2 Exo 3 
| . 
241 (18) 
224 (26) 
> 250 
A9PLJ1 
+ 
217 (28) 
159 (28) 
224 (21) 

± 
Z£12) + 
100 (351 
125 (29)+ 
‘All mice received IL-2 i.p. (15,000 u bid) for 4 days. Cell dose was 4-5 x 10 7 per mouse. 
-^Significant difference with no treatment and PLJ1 control groups (p < 0.05). 
We next examined the antitumor effect of these activated LN cells at larger doses. 
Utilizing the same protocol as Table 3, 7-13 x 10 7 activated cells were transferred into mice 
with established A9 parental lung metastases. As can be seen in Table 4, anti-CD3/IL-2 
activated cells derived from A94-3 draining LN resulted in >97% reduction of A9 pulmonary 
metastases in both experiments (Appendix C). 
Table 4: Immunotherapy of 3-day BL6 (A9) Pulmonary Metastases with Activated TDLN Cells 
From Mice Bearing IL-4 Transfected Tumor (A94-3) 
Source of 
TDLN 
Cells 
Transferred 3 
Mean No. of Pulmonary 
Metastases (SEM) 
Exp 1 Exp 2 
- 
- 
>250 
>250 
A9 
+ 
97 (16) b 
n.d. 

+ 
12 (Q) c 
Q b 
a No. of cells: Exp. 1 (7 x 10 7 ) and Exp. 2 (1.3 x 10 8 ). All mice received 15,000 u IL-2 i.p. bid 
for 5 days after cell transfer. 
b p < 0.05 compared to no cell treatment. 
c p < 0.05 compared to all other groups. 
From our previous animal studies, we had documented that the bacterial adjuvant, 
parvum . was useful in upregulating the in vivo sensitization of TDLN pre-effector cells against 
poorly immunogenic tumors (see Section 2.1). Utilizing this model, we compared the effect of 
C. parvum in eliciting sensitized pre-effector cells to A9 tumor compared with A94-3. Groups 
of B6 mice were inoculated s.c. with: A9PLJ1 tumor + C. parvum or A94-3 tumor + C. parvum . 
Recombinant DNA Research, Volume 18 
[791] 
