(1 1). This antibody has a variety of in vivo and in vitro immunomodulatory effects on T cells 
including the induction of nonspecific proliferation. Tumor-draining LN cells activated for 2 
days with anti-CD3 (1 ug/ml) alone did not result in significant proliferation. However, 
sequential activation with anti-CD3 followed by expansion in a low concentration of IL-2 (10 
u/ml) for 3 days resulted in approximately 10-fold increase in cell numbers (8). This 
proliferation was not specific to the tumor-draining LN cells because normal LN cells 
proliferated to an equivalent degree. In spite of this, our adoptive immunotherapy 
experiments revealed that the anti-CD3 activated tumor-draining, but not normal, LN cells 
had antitumor effects. 
Numerous reports have demonstrated that the in vitro stimulation of normal T cells with 
anti-CD3 results in proliferation and generation of cytotoxic effector cells (12,13). However, 
none of these anti-CD3 stimulated cells demonstrate antigen-specific reactivity probably due 
to the ubiquitous presence of CD3 on all T lymphocytes. Our experiments with anti-CD3/IL-2 
activation of tumor-draining LN cells suggested that specific stimulation of antigen-sensitized 
pre-effector cells had occurred. We therefore examined the specificity of adoptive 
immunotherapy mediated by anti-CD3/IL-2 activated cells. Anti-CD3/IL-2 activated MCA 106 
tumor-draining LN cells were transferred to mice with 3-day established metastases from 
three antigenically distinct murine sarcomas (Table 1). Mice lungs were harvested 14 days 
after tumor inoculation and metastases counted; too numerous to count tumors/lung were 
scored as > 250. There was significant antitumor reactivity against the MCA 106 tumor but 
not the MCA 102 or MCA 105 tumors. In separate experiments we found that similarly 
activated LN cells draining the MCA 105, MCA 203 or MCA 102 tumors mediated significant 
reductions of MCA 105, MCA 203 or MCA 102 metastases, respectively (data not shown) 
(ID- 
Table 1 : Specificity of Adoptive Immunotherapy Mediated by Anti-CD3/IL-2 Activated MCA 
106 Tumor-Draining Lymph Node Cells 
Treatment 
Mean no. pulmonary metastases (SEMI 
Anti-CD3 
IL-2 
activated 
(15000 u, ip 2x/day 
MCA 102 
MCA 105 
MCA 106 
cells(2x10 7 ) 
for 4 days) 
- 
- 
246 (4) 
>250 
>250 
- 
+ 
>250 
>250 
>250 
+ 
+ 
217(28) 
>250 
0‘ 
‘Compared to no treatment or IL-2 alone p <0.05 
Next, we examined the feasibility of generating anti-CD3/IL-2 activated effector cells 
reactive to the poorly immunogenic B16 BL6 melanoma. This subline of B16 melanoma, a 
spontaneously arisen tumor, is poorly immunogenic by virtue of the inability to elicit systemic 
immunity in syngeneic B6 mice experimentally. Additionally, therapeutically effective TIL 
cannot be generated from progressive subdermal tumors (B. Fox, data not shown). Initial 
experiments with the B16 BL6 tumor indicated that unlike the immunogenic MCA 106, 
therapeutic anti-CD3/IL-2 activated cells could not be induced from tumor-draining LN. 
However, it was possible to generate anti-CD3/IL-2 activated lymphocytes with therapeutic 
efficacy against the B16 BL6 tumor from LN draining an inoculum of tumor cells admixed with 
C. parvum (14,15). As noted in Table 2, the antitumor reactivity of tumor-primed anti-CD3/IL- 
2 activated LN cells was critically dependent upon the dose of bacterial adjuvant. Activated 
cells (5 x 10 7 /mouse) were transferred i.v. into mice with 3-day established B16 BL6 
Recombinant DNA Research, Volume 18 
[787] 
