Intraperitoneal injection of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of malignant ascites due to gastric cancer.

Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy-resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 µmol/L) and interleukin-2 (1000 IU/mL). After 14 days culture, Vγ9Vδ2 T-cells were harvested and administered intraperitoneally in four weekly infusions. The day before T-cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vγ9Vδ2 T-cell recognition. Seven patients were enrolled in this study. The number of Vγ9Vδ2 T-cells in each injection ranged from 0.6 to 69.8 × 10(8) (median 59.0 × 10(8)). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vγ9Vδ2 T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN-γ was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vγ9Vδ2 T-cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vγ9Vδ2 T-cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

Targeting spatiotemporal expression of CD137 on tumor-infiltrating cytotoxic T lymphocytes as a novel strategy for agonistic antibody therapy.

CD137 (4-1BB) is an important costimulatory ligand and a potent stimulator of T-cell responses. It has been used therapeutically to stimulate immunity against several solid malignancies as well as to modulate susceptibility to autoimmune disease and infection. However, clinical trials of anti-CD137 agonistic antibody have been suspended because of deleterious side effects. To overcome this problem, we fine-tuned the combination of adoptive transfer of tumor-specific cytotoxic T lymphocytes (CTL) and anti-CD137 monoclonal antibody (mAb) treatment. B16 melanoma cells (1 × 10(6)) were implanted subcutaneously in C57BL/6 mice. On day 9, CTLs (1 × 10(7)) were intravenously injected into tumor-bearing mice. Transferred CTL distributed throughout the body and infiltrated into the tumor. CD137 expression was upregulated on tumor-infiltrating CTL, but not in other tissues or other cell types. Therefore, mice received anti-CD137 mAb (100 µg) 3 days after CTL transfer. interferon-γ was produced in the tumor only by transferred CTL, not recipient-derived cells. The functional CTLs in the tumor were increased and interferon-γ production per cell was augmented by anti-CD137 treatment. It was not detected in CTL found in other tissues. Consistent with this, no organ damage was observed on anti-CD137 treatment. On the basis of the spatiotemporal expression of CD137 on tumor-infiltrating CTLs, anti-CD137 mAb selectively activated these tumor-infiltrating cells, augmented their antitumor activity and greatly decreased tumor growth. Tumor-specific CTL can guide agonistic anti-CD137 mAb to the tumor and in turn, become functionally augmented. Thus, CD137 mAb therapy may become feasible again in combination with tumor-specific CTL therapy.

A substantial incidence of silent short segment endoscopically suspected esophageal metaplasia in an adult Japanese primary care practice.

AIM: To determine the incidence and characteristics of endoscopically suspected esophageal metaplasia (ESEM) in a primary adult care institution. METHODS: Eight hundred and thirty two consecutive individuals (mean age, 67.6 years) undergoing upper gastrointestinal endoscopy between January 2009 and December 2010 were included in this study. The diagnosis of ESEM was based on the criteria proposed by the Japan Esophageal Society, and was classified as long segment ESEM (3 cm or more) or short segment ESEM (< 3cm). Short segment ESEM was further divided into circumferential and partial types. Age, gender, hiatus hernia, esophagitis, gastroesophageal reflux disease (GERD)-suggested symptoms, and antacid medications were recorded as background factors. Esophagitis was graded according to the Los Angeles classification. Hiatus hernia was divided into absent and at least partially present. RESULTS: Long and short segment ESEM were found in 0 and 184 (22.1%) patients, respectively (mean age of short segment ESEM patients, 68.3 years). Male gender and hiatus hernia were shown to be significant factors affecting short segment ESEM by both univariate (P = 0.03 and P = 9.9x10(-18)) and multivariate [Odds ratio (OR) = 1.45; P = 0.04, and OR = 43.3; P = 1.5x10(-7))] analyses. Two thirds of patients with short segment ESEM did not have GERD-suggested symptoms. There was no correlation between short segment ESEM and GERD-suggested symptoms. CONCLUSION: The incidence of short segment ESEM in our community practice seems higher than assumed in Asian countries. As GERD-suggested symptoms are a poor predictor of ESEM, endoscopists should bear in mind that silent short segment ESEM does exist and, in fact, was found in the majority of our patients.

Efficient cross-presentation of soluble exogenous antigens introduced into dendritic cells using a weak-based amphiphilic peptide.

To develop a novel dendritic cell (DC)-based vaccine for inducing antigen-specific CD8+ T cell responses by cross-presentation, we tested a novel antigen delivery system that introduces soluble antigens into the cytosol of cells by an endocytosis-mediated mechanism which avoids damaging the plasma membrane ("Endo-Porter"). Proteins released from endosomes into the cytoplasm are degraded by the proteasome, and fragmented antigenic peptides are presented to the classical cytosolic MHC class I pathway. DCs pulsed with OVA protein in the presence of Endo-Porter efficiently stimulate OVA peptide-specific CD8+ T (OT-I) cells. Although this agent diverts some of the endocytosed antigens away from the classical MHC class II-restricted presentation pathway to the class I pathway, the activation of CD4+ T cells was found not to be hampered by Endo-Porter-mediated antigen delivery. On the contrary, it was rather augmented, probably due to the increased uptake of antigen. Because specific CD4+ T cell help is required to license DCs for cross-priming, Endo-Porter-mediated antigen delivery is a promising approach for developing more efficient cancer vaccines targeting both CD4+ and CD8+ T cells.

Impact of culture medium on the expansion of T cells for immunotherapy.

BACKGROUND AIMS: Encouraging evidence of clinical benefits from cancer immunotherapy is beginning to accumulate in several clinical trials. Cancer immunotherapy is based on two main methods, active vaccination and cell-transfer therapy. The ex vivo expansion of T cells is required to monitor vaccine-induced antigen-specific T cells or prepare large numbers of reactive lymphocytes for adoptive transfer. METHODS: We examined the influence of culture medium on T-cell growth, cytotoxicity and phenotype after activation using immobilized anti-CD3 monoclonal antibody or Zoledronate stimulation. Peripheral blood mononuclear cells (PBMC) were cultured in RPMI, AIM-V or OpTmizer with or without autologous serum. RESULTS: When supplemented with sufficient serum, RPMI was a good culture medium for T-cell expansion following anti-CD3 stimulation. Addition of autologous serum to AIM-V or OpTmizer increased the numbers of cells obtained to a similar extent, but their phenotype and function were quite different. Activated T cells cultured with OpTmizer mediated greater cytotoxicity than any other culture. Regardless of the media used, the main population expanded after CD3 stimulation was CD3(+) CD8(+). While more CD3(+) CD4(+) T cells were induced in RPMI and AIM-V, more CD3(-) CD56(+) cells and CD3(+) CD56(+) T cells were induced in OpTmizer. When cells were stimulated by Zoledronate for 14 days, approximately 7.2 times and 11.5 times more gammadelta T cells were obtained in OpTmizer than AIM-V or RPMI, respectively. CONCLUSIONS: Successful immunotherapy depends on the selection of appropriate culture media to support efficient expansion of the type of T cell desired.