Cytokine gene-modified tumor cells have increased immunogenicity and retain the antigenic repertoire of a particular neoplasia. However, practical concerns have led to an increased interest in allogeneic gene-transduced bystander cells as a broader source of cytokines for autologous tumor cell-based vaccines. Here, we show that allogeneic B78H1 major histocompatibility complex (MHC) class I-negative and -positive (H-2K(b)- and D(b)-transfected) cells induced cytotoxic T lymphocytes (CTLs) and protection in BALB/c mice at comparable levels in response to a challenge with C26 (H-2(d)) colon carcinoma cells sharing the tumor-associated antigen envelope glycoprotein 70 (env-gp70) with both cell lines. Class I-negative B78H1 cells transduced to express interleukin-12 (IL-12) and mixed with autologous A20 tumor cells led to eradication of preestablished A20 lymphoma in 50% or 100% of treated mice after 3 or 4 vaccinations, respectively, whereas A20 cells alone or mixed with nontransduced B78H1 cured none or 50% of mice after 3 or 4 vaccinations, respectively. Immunization with the IL-12-producing bystander cell line increased tumor-specific proliferation and type 1 cytokine production by CD4(+) T cells. By contrast, CD4 T-cell function appeared impaired after immunization with A20 cells alone or mixed with B78H1 cells. Indeed, only CD4(+) T cells from IL-12-treated mice could be restimulated with anti-OX40 monoclonal antibody (mAb) in place of a fourth cellular boost. Moreover, the IL-12-based tumor vaccine induced expansion of tumor-specific interferon-gamma (IFN-gamma)-producing CD8(+) T cells. These results are clinically relevant for the development of feasible IL-12 cancer vaccines based on engineered class I-negative bystander cells.