Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3­specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

Effects of food lectins on the transport system of human intestinal Caco-2 cell monolayers.

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20-40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.

Copulsing tumor antigen-pulsed dendritic cells with zoledronate efficiently enhance the expansion of tumor antigen-specific CD8+ T cells via Vgamma9gammadelta T cell activation.

We demonstrate that Vgamma9gammadelta T cells activated by zoledronate can link innate and acquired immunity through crosstalk with dendritic cells (DCs) in a way that can amplify activation and proliferation of tumor antigen-specific CD8+ T cells. DCs pulsed with antigen alone or antigen plus zoledronate were used to stimulate the in vitro expansion of antigen-specific CD8+ T cells. MART-1-modified peptide (A27L peptide) and apoptotic HLA-A*0201-positive, MART-1-positive JCOCB tumor cell lines were used as tumor antigen sources. The percentage of A27L-specific CD8+ T cells within the responding lymphocytes on Day 7 when immature DCs (imDCs) were cultured in the presence of A27L peptide and 0.01 microM zoledronate was significantly higher (P=0.002, n=11) than that observed when imDCs were cultured with the lymphocytes in the presence of the A27L peptide alone. This enhancing effect of zoledronate was significantly reduced when gammadelta T cells were depleted from responding lymphocytes (P=0.030, n=5), indicating that the effect is mediated mainly through Vgamma9gammadelta T cells activated by zoledronate-pulsed imDCs. When imDCs copulsed with zoledronate and apoptotic JCOCB tumor cell lines were used, the percentage of A27L-specific CD8+ T cells was higher than that observed using imDCs with the apoptotic JCOCB lines alone, suggesting that zoledronate treatment of imDCs enhances the cross-presentation ability of DCs. These findings suggest a potentially valuable role for Vgamma9gammadelta T cell activation for expanding antigen-specific CD8+T cells using DCs copulsed with tumor antigen and zoledronate in the design of vaccine therapies for malignancy.

Mature dendritic cells are superior to immature dendritic cells in expanding antigen-specific naive and memory CD8+ T cells.

BACKGROUND: For successful dendritic cell (DC)-based immunotherapy, it is critical to identify the most potent stage of human DCs, including immature DCs (imDCs) and mature DCs (mDCs). MATERIALS AND METHODS: imDCs were obtained by culturing monocytes in the presence of GM-CSF and IL-4 for 5- 7 days and imDCs were further cultured for 24-48 h in the presence of TNFalpha, IL-6, IL-1beta and PGE2 to obtain mDCs. Melan-A- and EBV (BRF1) peptides were used and the frequency of antigen-specific CD8+ T cells was assessed using appropriate tetramers. RESULTS: mDCs were potent antigen-presenting cells for the induction and proliferation of antigen-specific naive and memory CD8+ T cells and may overcome regulatory functions that suppress antigen-specific CD8+ T cells. CONCLUSION: Our findings that mDCs can efficiently expand antigen-specific naive and memory CD8 + T cells have important implications in the development of vaccination strategies and support the use of antigen-loaded mature DCs in human clinical trials

Ex vivo enhancement of antigen-presenting function of dendritic cells and its application for DC-based immunotherapy.

Dendritic cells (DCs) are potent antigen presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. In particular DCs generated from peripheral blood monocytes (Mo-DCs) have been used with promising results as a new approach for the immunotherapy of cancer. In this study, we have analyzed the changes in the pattern of expression molecules on Mo-DCs during DC maturation using different maturation cytokine combinations and the expansion capacity of an antigen specific CD8+T cells monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. These analyses revealed that the expansion of antigen specific CD8+T cells is the most effective when T cells were activated by fully maturated DCs by culturing monocytes for 5 days in the presence of GM-CSF and IL-4, followed by 2-3 days of maturation with pro-inflammatory mediators including TNFalpha, IL-6, IL-1beta and PGE2. These results pave the way to a more effective immunotherapy using DCs for patients with malignancy, as well as infectious diseases.