[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) can be differentiated from CD14+ monocytes in the presence of interferon-α (IFNα) and granulocyte/macrophage colony stimulating factor (GM-CSF) in vitro, and are known as IFN-DCs. Circulating blood CD56+ cells expressing high levels of CD14, HLA-DR, and CD86 have been shown to spontaneously differentiate into DC-like cells in vitro after their isolation from blood. We show here that IFN-DCs expressing high levels of CD56 (hereafter, CD56high+IFN-DCs) can be differentiated in vitro from monocytes obtained as adherent cells from healthy donors and patients with metastatic melanoma. These cells expressed high levels of CD14, HLA-DR, and CD86, and possessed many pseudopodia. These CD56high+IFN-DCs may be an in vitro counterpart of the circulating CD56+CD14+CD86+HLA-DR+ cells in blood. Conventional mature DCs differentiated from monocytes as adherent cells in the presence of GM-CSF, IL-4, and TNF-α (hereafter, mIL-4DCs) did not express CD56 or CD14. In contrast to mIL-4DCs, the CD56high+IFN-DCs exhibited a stronger capacity to stimulate autologous CD56+Vγ9γδT cells highly producing IFNγ in the presence of zoledronate and IL-2. The CD56high+IFN-DCs possessing HLA-A*0201 effectively induced Mart-1-modified melanoma peptide (A27L)-specific CD8+ T cells through preferential expansion of CD56+Vγ9γδT cells in the presence of A27L, zoledronate, and IL-2. Vaccination with CD56high+IFN-DCs copulsed with tumour antigens and zoledronate may orchestrate the induction of various CD56+ immune cells possessing high effector functions, resulting in strong immunological responses against tumour cells. This study may be relevant to the design of future clinical trials of CD56high+IFN-DCs-based immunotherapies for patients with melanoma.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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 GPC3‑specific 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.
International Journal of Oncology 09/2014; 45(5). DOI:10.3892/ijo.2014.2634 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adoptive immunotherapy of cancer is evolving with the development of novel technologies for generating a large number of activated killer cells such as natural killer (NK) cells, γδ T cells, and αβ T cells. We have recently established large-scale culture methods to generate activated NK cells from human peripheral blood, and demonstrated that expanded NK cells have higher cytotoxicity against cancer cells than freshly isolated NK cells. In this study, we compared cultured NK cells with cultured γδ T and αβ T cells that were prepared by conventional culture methods regarding the expression of cytotoxic molecules and cytotoxicity against cancer cells. Natural cytotoxicity receptors such as NKp30, NKp44 and NKp46, and perforin were expressed most exclusively on NK cells. Granzyme A, NKG2D, and interferon-γ were dominantly expressed in NK cells and γδ T cells but not in αβ T cells. Consistent with the expression profiles of the cytotoxic molecules, cultured NK cells from both healthy volunteers and cancer patients demonstrated significantly higher cytotoxicity against cancer cell lines, including MHC class I-positive cell lines, compared with cultured γδ T cells and cultured αβ T cells. Additionally, NK cells, unlike γδ T cells or αβ T cells, expressed high levels of CD16, and showed augmented cytotoxicity when co-administered with an anti-CD20 monoclonal antibody drug, rituximab. These results suggest the excellent efficacy of expanded NK cells for cancer treatment.
International Immunopharmacology 08/2014; 22(2):486-491. DOI:10.1016/j.intimp.2014.07.036 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A potential adverse effect of dipeptidyl peptidase-4 inhibitors (DPP-4i) on the pancreas remains controversial. We evaluated the DPP-4i effects on pancreatic amylase and lipase activity in patients with type 2 diabetes. These enzymes were slightly but significantly increased, suggesting DPP-4i cause a low-grade inflammatory change in the exocrine pancreas.
Diabetes research and clinical practice 04/2013; 100(3). DOI:10.1016/j.diabres.2013.03.034 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent commercial approval of cancer vaccine, demonstrating statistically significant improvement in overall survival of prostate cancer patients has spurred renewed interest in active immunotherapies; specifically, strategies that lead to enhanced biological activity and robust efficacy for dendritic cell vaccines. A simple, widely used approach to generating multivalent cancer vaccines is to load tumor whole cell lysates into dendritic cells (DCs). Current DC vaccine manufacturing processes require co-incubation of tumor lysate antigens with immature DCs and their subsequent maturation. We compared electroloading of tumor cell lysates directly into mature DCs with the traditional method of lysate co-incubation with immature DCs. Electroloaded mature DCs were more potent in vitro, as judged by their ability to elicit significantly (p<0.05) greater expansion of peptide antigen-specific CD8+ T cells, than either lysate-electroloaded immature DCs or lysate-co-incubated immature DCs, both of which must be subsequently matured. Expanded CD8+ T cells were functional as judged by their ability to produce IFN-γ upon antigen-specific re-stimulation. The electroloading technology used herein is an automated, scalable, functionally closed cGMP-compliant manufacturing technology supported by a Master File at CBER, FDA and represents an opportunity for translation of enhanced potency DC vaccines at clinical/commercial scale.
International immunopharmacology 02/2013; 15(3):488-497. DOI:10.1016/j.intimp.2013.01.009 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The adoptive transfer of highly cytotoxic natural killer (NK) cells is an emerging tool for cancer immunotherapy. Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical factors for the clinical efficacy of anticancer antibodies, in which NK cells are the major effectors of ADCC. NK cells were expanded from PBMC by a feeder-cell-free expansion method. NK cell expansion efficiency was evaluated within a period of 21days. The kinetics of NK cell expansion and the expression of activating and inhibitory receptors on NK cells were monitored. NK cells producing IFN-γ and TNF-α were detected by intracellular cytokine staining. The cytotoxicity of expanded NK cells against various cancer cells was compared with that of freshly isolated NK cells. The ADCC functions of expanded NK cells in combination with rituximab against CD20+ lymphoma cell lines were evaluated. Our method efficiently expanded NK cells ex vivo, which showed a much higher activity to induce the expression of activating receptors and to produce IFN-γ and TNF-α as well as cytotoxicity against various cancer cell lines including CD133+ primary cancer cells than freshly isolated NK cells. We observed a synergistic cytotoxicity of our expanded NK cells against CD20+ B lymphoma cell lines as well as higher IFN-γ and TNF-α production when combined with rituximab. Our results suggest that the adoptive transfer of a large number of ex vivo expanded NK cells, particularly in combination with monoclonal antibody drugs, is a useful tool for cancer immunotherapy.
International immunopharmacology 10/2012; 14(4):593-605. DOI:10.1016/j.intimp.2012.09.014 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adoptive transfer of ex vivo expanded autologous Vγ9Vδ2 T cells may be of therapeutic benefit for cancer because of their potent direct cytotoxicity towards tumour cells, synergistic cytotoxicity when combined with aminobisphosphonates and enhancement of antibody-dependent cell-mediated cytotoxicity.
To determine the feasibility and clinical safety of therapy with ex vivo expanded, activated Vγ9Vδ2 T cells in combination with zoledronate, we enrolled 18 subjects with advanced solid tumours into a phase I clinical study. Administered indium(111)-oxine-labelled Vγ9Vδ2 T cells were tracked in a cohort of patients.
Administered Vγ9Vδ2 T cells had an activated effector memory phenotype, expressed chemokine receptors predictive of homing to peripheral tissues and were cytotoxic in vitro against tumour targets. Adoptively transferred Vγ9Vδ2 T cells trafficked predominantly to the lungs, liver and spleen and, in some patients, to metastatic tumour sites outside these organs. No dose-limiting toxicity was observed, but most patients progressed on study therapy. However, three patients administered Vγ9Vδ2 T cells while continuing previously ineffective therapy had disease responses, suggesting an additive effect.
Therapy with aminobisphosphonate-activated Vγ9Vδ2 T cells is feasible and well tolerated, but therapeutic benefits appear only likely when used in combination with other therapies.
British Journal of Cancer 08/2011; 105(6):778-86. DOI:10.1038/bjc.2011.293 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human Vα24+Vβ11+ natural killer T-cells (NKT cells) have antitumor activity via direct cytotoxicity and by induction of antitumor actions of T and NK cells. Activation of NKT cells is crucial for their antitumor activity and is induced by α-galactosylceramide (α-GalCer, KRN7000) presented by CD1d on dendritic cells (DC). We conducted a phase I clinical trial of therapy with α-GalCer-pulsed DC to determine safety, tolerability, immune effects and an optimal dose, and administration route.
Twelve subjects (3 cohorts) with metastatic malignancy received 4 treatments of α-GalCer-pulsed DC, 2 treatments intravenously (IV), and 2 treatments intradermally (ID). Each successive cohort received a log higher cell dose. Clinical and immunological outcomes were evaluated, including secondary effects on NK and T cells.
Substantial effects on peripheral blood NKT cells were observed but were greater following IV treatment. Secondary immune effects including activation of T and NK cells, increases in T- and NK-cell cytoplasmic interferon-γ, and increases in serum interferon-γ levels were seen after IV but not after ID treatment. Therapy was well tolerated, but 9 of 12 subjects had tumor flares with clinical findings consistent with transient tumor inflammation. Disease response (minor) or stabilization of disease progressing up to enrollment was observed in 6 of the 12 subjects. Stabilization of previously progressive disease lasted for at least one year in three subjects.
We conclude that therapy with α-GalCer-pulsed DC induced clinically beneficial immune responses that are highly dependent on cell dose and administration route.
Clinical Cancer Research 06/2011; 17(15):5140-51. DOI:10.1158/1078-0432.CCR-10-3105 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cell (DC) therapy frequently induces a measurable immune response. However clinical responses are seen in a minority of patients, presumably due to insufficient expansion of antigen-specific cytotoxic T lymphocytes (CTLs) capable of eradicating tumor cells. To increase therapeutic efficacy of DC-based vaccination, we have undertaken the first clinical trial involving a combination therapy of gemcitabine (GEM) with immunotherapy for patients with inoperable locally advanced pancreatic cancer.
Patients (n = 5) received the treatment course, which consisted of intravenous GEM administration at 1000 mg/m (day 1) and the endoscopic ultrasound-guided fine-needle injection of OK432-pulsed DCs into a tumor, followed by intravenous infusion of lymphokine-activated killer cells stimulated with anti-CD3 monoclonal antibody (CD3-LAKs) (day 4), at 2-week intervals.
No serious treatment-related adverse events were observed during the study period. Three of the 5 patients demonstrated effective responses to this clinical trial; 1 had partial remission and 2 had long stable disease more than 6 months. In the patient with partial remission, it has been shown that DC-based vaccination combined with GEM administration induces tumor antigen-specific CTLs.
This combined therapy was considered to be synergistically effective and may have a role in the therapy of pancreatic cancer for inducing tumor antigen-specific CTLs.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the potential anti-tumor activity of zoledronate-activated Vgamma9gammadelta T cells in vivo, we initiated a pilot study involving administration of zoledronate-activated Vgamma9gammadelta T lymphocyte-activated killer (LAK) cells to patients with multiple myeloma.
Subjects (n = 6) received four intravenous infusions at 2-week intervals of zoledronate-activated Vgamma9gammadelta T LAK cells generated from the culture of peripheral blood mononuclear cells (PBMCs) in the presence of zoledronate and interleukin-2. If the M-protein level in the patient's serum remained at baseline following four intravenous infusions, the patient underwent four more treatments at 4-week intervals. Subjects (n = 6) received a median of 0.99 x 10(9) Vgamma9gammadelta T LAK cells per infusion.
No serious treatment-related adverse effects were observed during the study period. The percentage of Vgamma9gammadelta T cells in PBMCs and absolute numbers of Vgamma9gammadelta T cells in peripheral blood, particularly those of CD45RA(-)CD27(-) effector memory (TEM) Vgamma9gammadelta T-cell subsets increased in all the patients. Percentages of Vgamma9gammadelta T cells and TEM Vgamma9gammadelta T cells in bone marrow also increased in all the patients so far tested. M-protein levels in the serum remained at baseline in four of six patients and increased in two of six patients. Soluble major histocompatibility complex class I chain-related antigen A was detected only in the serum of patients whose M-protein level increased.
Administration of zoledronate-activated Vgamma9gammadelta T LAK cells is a safe and promising immunotherapy approach for treatment of patients with multiple myeloma.
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms of HLA-A, B, C, DR, and DQ antigens were investigated in a Mongoloid population named Buryat living in Siberia. HLA gene and haplotype frequencies were calculated from the population data obtained from 141 unrelated healthy Buryat adults. Gene frequencies of class I antigens A2, A24, A1, B61, Cw10, and Cw6 were estimated to be more than 10%. For class II, DR4, DR7, DR13, DQ7, and DQ1 antigens were predominant. A phylogenetic tree was constructed based on HLA gene frequencies, and the Buryat population was clustered with the Mongoloid groups in Northeast Asia. In the analysis of HLA-A, C, B, DR, and DQ five-locus haplotype frequencies, seven kinds of haplotypes were calculated to occur at frequencies of more than 2%. Five of the seven common haplotypes have also been described in the other human populations thus far. Some of the haplotypes have been described in European populations, while the others were shared with Northeast Asian Mongoloids as well as Amerindians. Similar situation was also found in the analysis of class I (HLA-A, C, B) three locus haplotypes. These observations suggest the unique genetic background of this Buryat population.