The
colon adenocarcinoma C26, carrying two endogenous
tumor-associated
antigens (TAA) recognized by CTL, has been transduced with the gene coding for the human
folate receptor alpha (FR alpha) as an additional
antigen in order to study the efficacy of vaccination against a
tumor expressing multiple
antigens. A dicistronic vector was used to transduce the
IL-12 genes to create C26/
IL-12/FR alpha that has been used as a cellular
vaccine to treat mice bearing lung
metastases of C26/FR alpha. After vaccination mice were partially splenectomized and splenic lymphocytes frozen and used retrospectively to study in vitro CD8 T cell response related to the treatment outcome. Vaccination cured 50% of mice and the effect was CD8 T cell dependent. Mice either cured (responders) or not cured (nonresponders) by vaccination developed
tumor-specific CTL. However, analysis of CTL specificity and pCTL frequencies revealed that responders had a predominant CTL activity against endogenous C26-related
tumor antigens, whereas nonresponders had CTL that recognized preferentially the FR alpha
antigen. CD8 from responder mice were characterized to release high levels of granulocyte-macrophage (
GM)-CSF upon
antigen stimulation.
Tumors obtained from mice that died despite vaccination lost expression of the FR alpha transgene but maintained expression of endogenous C26
antigens. Immunoselection against FR alpha
antigen was not observed in
tumors from non-vaccinated controls and from CD8-depleted vaccinated mice. Down-regulation of FR alpha
antigen expression was due, at least in part, to methylation of retroviral vector long terminal repeat promoter since FR alpha expression was partially restored, ex vivo, by treatment with 5-aza-2'-deoxy-cytidine (aza). These results indicate that CD8 T cell-mediated immunoselection and production of
GM-CSF are determining factors for the efficacy of
tumor vaccines.