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In an effort to establish cytolytic T lymphocytes (CTLs) against colorectal carcinoma (CRC) by stimulating patients' lymphocytes with autologous tumor cells, we used peripheral blood mononuclear cells (PBMC) from a patient with minimal residual rectal carcinoma following removal of the primary lesion and involved regional lymph nodes as a source to generate CTLs in culture. A CTL line and clone were established from the patient's PBMC following stimulation of PBMC with autologous, cultured tumor cells and interleukin-2. The CTL line and the clone consisted predominantly of CD4+ lymphocytes. The CTL clone expressed two T-cell receptor variable alpha chains (V alpha11 and V alpha22) and one beta chain (Vbeta14). The cytokine secretion pattern of the CTL line was of the Th1-type. Both the CTL line and the clone lysed the autologous rectal carcinoma cells, but not the allogeneic, partially human lymphocyte antigen (HLA)-matched or nonmatched CRC cells, autologous Epstein-Barr virus-transformed B cells, K562 (natural killer target) cells or Daudi (lymphokine-activated killer target) cells. Lysis of autologous tumor cells most likely was HLA class I-restricted. Our unique success in generating CTLs against this tumor type may rest in the inclusion of a patient with minimal residual, rather than advanced, disease.

The human colorectal carcinoma ( CRC ) -associated GA733 antigen ( Ag ), also named CO17 -1A / EpCAM / KSA / KS1 -4, has been a useful target in passive immunotherapy of CRC patients with monoclonal antibody ( mAb ) and in active immunotherapy with antiidiotypic antibodies or with recombinant protein. These approaches have targeted single epitopes ( monoclonal anti -GA733 antibodies and anti -idiotypic antibodies ) or extracellular domain epitopes ( recombinant protein ), primarily by B cells. To determine whether a reagent that induces immunity to a larger number of both B -and T -cell epitopes might represent a superior vaccine, we analyzed the capacity of full -length GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant vaccinia virus ( VV GA733 -2 ) to induce humoral, cellular, and / or protective immunity in mice. VV GA733 -2 induced Ag -specific antibodies that reacted predominantly to unknown epitopes on the Ag and lysed Ag -positive CRC targets in conjunction with murine peritoneal macrophages as effector cells. Immunized mice developed Ag -specific, proliferative and delayed -type hypersensitive lymphocytes. VV GA733 -2 inhibited growth of ras -transformed syngeneic tumor cells expressing the human GA733 Ag in mice. These results suggest the potential of VV GA733 -2 as a candidate vaccine for patients with CRC, possibly in combination with recombinant GA733 -2 -expressing adenovirus, which has been shown to induce cytolytic antibodies and T cells as well as tumor protective effects in mice. The combined vaccine approach may be superior to the use of either vaccine alone in patients who are pre -immune to both viruses.

Mice lacking functional T and B lymphocytes offer an in vivo animal model for the study of human immune functions. We have attempted to optimize the reconstitution of severe combined immunodeficiency (SCID) mice with human peripheral blood lymphocytes (PBL) using radiation, anti-asialo GM1 antibody or cyclophosphamide (Cy) treatment of the mice and in vitro stimulation of human PBL with interleukin (IL)-2 prior to their transfer to the mice. Total human IgG and tetanus-toxoid (TT)-specific human IgG responses of the mice were used as parameters of successful reconstitution. Treatment of the mice with anti-asialo GM1 antibody significantly enhanced total human IgG levels, but not TT-specific antibody responses, whereas irradiation or Cy treatment of the mice had no effect on human antibody production. In vitro treatment of human PBL with IL-2 prior to engraftment significantly decreased total human IgG responses of human PBL-grafted SCID mice. The immune responses of individual mice within a group were highly variable, which constitutes a major disadvantage of this model.

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