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We have conducted a pilot study with combined immunotherapy using autologous lymphocytes activated ex vivo and monocyte-derived dendritic cells in combination with low-dose OK-432, a streptococcal preparation, in five patients with peritoneal or pleural carcinomatosis who were resistant to standard chemotherapy. All patients were given 3 to 10 courses of the combined immunotherapy. No severe adverse reactions occurred. Effusion production was decreased in all of the patients. Significant decreases in tumor markers of both effusions and sera as well as effusion volume occurred in all of the patients. Cytological examinations revealed a marked decrease or disappearance of cancer cells in those effusions. Three patients showed increase in IFN-gamma levels in the effusions. The overall prognosis of the patients was acceptable and the mean survival time was more than 9 months. The locoregional immunotherapy seems to be encouraging in view of therapeutic modality in patients who are resistant to standard chemotherapy. Our study provides a new protocol for immunotherapy and warrants further phase I/II clinical study for chemo-resistant patients with malignant effusion.

Background & Aims: Previous work has suggested that CD4+ CD28- or costimulation-independent T cells are increased in autoimmune diseases. In this study, we compared frequency and qualitative characteristics of autoreactive costimulation-independent or CD4+ CD28- T cells in primary biliary cirrhosis (PBC) by taking advantage of the well-defined immunodominant autoepitope of the E2 component of pyruvate dehydrogenase (PDC-E2)., Methods: We determined the frequency of costimulation-independent autoreactive T cells that respond to PDC-E2 163-176 and the frequency of CD4+ CD28- T cells. Finally, we determined the role of biliary epithelial cells (BEC) as both an antigen-presenting cell or, alternatively, as a target cell for T-cell-mediated cytotoxicity., Results: The precursor frequency of costimulation-independent CD4+ T cells that respond to PDC-E2 163-176 and the frequency of CD4+ CD28- T cells were dramatically elevated in PBC. Furthermore, 2 types of T-cell clones that respond to PDC-E2 163-176 emerged from this study. One type was costimulation dependent and the other costimulation independent. Both types of clones lyse BEC in a similar effector target (E/T) ratio distribution. However, BEC did not help the proliferation of any T-cell clones. Furthermore, costimulation-independent T-cell clones do not become anergic by BEC., Conclusions: In PBC, costimulation-independent autoreactive T cells, which do not become anergic, increase and maintain the autoimmune response. In controls, although autoantigens are expressed on BEC and autoantigen-reactive T cells exist around BEC, autoantigen-reactive T cells are costimulation dependent and will become anergic and maintain peripheral tolerance.

Clinical use of Herceptin (trastuzumab), which is a humanized monoclonal antibody against HER2, started for patients with HER2-overexpressing breast cancer. To potentiate the efficacy of the Herceptin therapy, this study focused on the combination of Herceptin with activated immune lymphocytes. We used peripheral blood mononuclear cells (PBMCs) as effector cells and used HER2-unexpressing K562 cells, HER2-weakly-expressing breast carcinoma cells (Breast-M), or HER2-strongly-expressing breast carcinoma cells (BT-474) as target cells. Interleukin-2 (IL-2)-activated PBMCs, IL-2/OKT-3-activated PBMCs and a streptococcal preparation OK-432-activated PBMCs were generated and used as effector cells. Cytotoxic activity was determined with 4-hour 51Cr release assay. Both fresh PBMCs and activated PBMCs exhibited Herceptin-dependent cytotoxicity. Importantly, Herceptin-dependent cytotoxicity was found even at a lower effector to target cell ratio (E/T ratio) than that of Herceptin-independent cytotoxicity. In addition, Herceptin-dependent cytotoxicity by these activated PBMCs was observed even in HER2-weakly-expressing Breast-M cells. Since gamma-globulin or anti-CD16 antibody abrogated Herceptin-dependent cytotoxicity, it seems likely that antibody-dependent cellular cytotoxicity (ADCC) plays an important role in the Herceptin-dependent cytotoxicity. We present a recurrent breast cancer patient with malignant pleural effusion, in which HER2-strongly-expressing tumor cells were present, who was undergoing Herceptin therapy. Cluster formation between tumor cells and intrapleural mononuclear cells was induced 24 hours after intravenous injection of Herceptin (4 mg/kg). Mononuclear cells bound specifically to HER2-strongly-expressing tumor cells but not to other cells, such as mesothelial cells, suggested a Herceptin-mediated binding like ADCC in vivo. Taken together, these findings suggest that the combination of Herceptin with various types of activated lymphocytes may be a new therapeutic strategy, not only for HER2-strongly-expressing breast cancer but also for HER2-weakly-expressing cancer.

For vaccinations based on dendritic cells (DCs), maturation of DCs is critical to the induction of T-cell responses. We tested the efficacy of streptococcal preparation OK-432 as a Good Manufacturing Practice (GMP)-grade maturation agent. OK-432 is currently used in Japan as a cancer immunotherapy drug. Immature monocyte-derived dendritic cells (imMo-DCs) isolated from human peripheral blood monocytes stimulated with granulocyte-macrophage colony stimulating factor and interleukin-4 were exposed to maturation factors, i.e., lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 (PGE2), and OK-432 for 2 days. OK-432 increased expression of activation- and maturation-related molecules such as HLA-DR, CD80, CD83, and CD86 in imMo-DCs at levels similar to that of TNF-alpha plus PGE2, and higher than that of LPS. All agents examined induced allogeneic T-cell proliferation at a similar level. Only OK-432 caused significant production of interleukin-12 (IL-12) p70 and interferon gamma (IFN-gamma) at both the mRNA and protein levels in imMo-DCs. Neutralizing antibody against IL-12 p70 blocked IFN-gamma secretion from OK-432-stimulated Mo-DCs. IL-12 p70 produced by OK-432-stimulated imMo-DCs induced secretion of IFN-gamma by CD4+ T cells. OK-432 and LPS activated nuclear factor kappa B (NF-kappaB) in imMo-DCs. Both secretion of IL-12 p70 and IFN-gamma and activation of NF-kappaB induced by OK-432 were suppressed when imMo-DCs were pretreated with cytochalasin B. These results indicate that uptake of OK-432 by imMo-DCs is an early critical event for IL-12 p70 production and that NF-kappaB activation induced by OK-432 also contributes partially to IL-12 p70 production. In conclusion, OK-432 is a GMP-grade maturation agent and may be a potential tool for DC-based vaccine therapies.

Dendritic cells (DCs) are antigen-presenting cells specialized for the induction of the primary T-cell response. Tumor immunotherapy using DCs loaded with tumor antigens is under way for patients with several types of advanced malignancies. In this study, DC-like cells (Mo-DCs) were generated from peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The antigen-presenting abilities, including capture of apoptotic tumor cells, IL-12 secretion, expression of antigen-presentation-related molecules (HLA-ABC, HLA-DR, and CD80), and mixed leukocyte reaction, of Mo-DCs from 37 patients with advanced cancer (pMo-DCs) were compared to those of 20 healthy volunteers (hMo-DCs). Seven days after the initial culture, no significant difference was found in either the number or the size of Mo-DC-forming colonies between the two groups. However, most of the antigen-presenting abilities of pMo-DCs were weaker than those of hMo-DCs on day 7. On day 14, both number and size of colonies were significantly decreased in pMo-DCs but not in hMo-DCs. Interestingly, the antigen-presenting abilities of the remaining pMo-DCs gradually strengthened with time and by day 14 no significant difference was observed between pMo-DCs and hMo-DCs. These results indicate that pMo-DCs contain dysfunctional and short-lived Mo-DC subsets.