Maintenance therapy with ex vivo expanded lymphokine-activated killer cells and rituximab in patients with follicular lymphoma is safe and may delay disease progression.

Anti-cluster of differentiation 20 (CD20) monoclonal antibodies (mAbs) have shown promise in follicular lymphoma (FL) as post-induction therapy, by enhancing antibody-dependent cellular cytotoxicity (ADCC). However, cytotoxic cells are reduced after this treatment. We hypothesised that ex vivo expanded lymphokine-activated killer (LAK) cells administered to FL-remission patients are safe and improve anti-CD20 efficacy. This open, prospective, phase II, single-arm study assessed safety and efficacy of ex vivo expanded LAK cells in 20 FL-remission patients following rituximab maintenance. Mononuclear cells were obtained in odd rituximab cycles and stimulated with interleukin 2 (IL-2) for 8 weeks, after which >5 × 108 LAK cells were injected. Patients were followed-up for 5 years. At the end of maintenance, peripheral blood cells phenotype had not changed markedly. Natural killer, LAK and ADCC activities of mononuclear cells increased significantly after recombinant human IL-2 (rhIL-2) stimulation in all cycles. Rituximab significantly enhanced cytotoxic activity. No patients discontinued treatment. There were no treatment-related serious adverse events. Three patients had progressed by the end of follow-up. After a median (interquartile range) follow-up of 59.4 (43.8-70.9) months, 85% of patients remained progression free. No deaths occurred. Quality-of-life improved throughout the study. Post-induction LAK cells with rituximab seem safe in the long term. Larger studies are warranted to confirm efficacy.

Cell transfer-based immunotherapies in cancer: A review.

In cell transfer therapy (CTT), immune cells such as innate immune-derived natural killer cells and dendritic cells as well as acquired immune-related T lymphocytes such as tumor-infiltrating lymphocytes and cytokine-activated or genetically modified peripheral blood T cells are used in the management of cancer. These therapies are increasingly becoming the most used treatment modality in cancer after tumor resection, chemotherapy, and radiotherapy. In adoptive cell transfer, the lymphocytes isolated from either a donor or the patient are modified ex vivo and reinfused to target malignant cells. Transferring in vitro-manipulated immune cells produces a continuous antitumor immune response. In this review, we evaluate the recent advances in CTT for the management of various malignancies.

Harnessing Natural Killer Cell Antitumor Immunity: From the Bench to Bedside.

Natural killer (NK) cells are critical effector lymphocytes mediating tumor immune surveillance and clearance. They do so by direct tumor killing using cytolytic granules and death receptors, and by interfacing with and potentiating adaptive immune responses through the production of cytokines. From a therapeutic perspective, NK cells have been shown to exert graft-versus-leukemia activity in the context of hematopoietic stem cell transplantation and are important in the clinical efficacy of antibodies. Advances in basic and translational NK cell biology have led to multiple potential strategies to augment their in vivo activity to improve antitumor responses. Despite their potent effects, NK cells have been shown to be safe for adoptive cell therapy in both the autologous and allogeneic settings, with promising, but so far limited, clinical efficacy. This review will provide an overview of strategies being pursued to improve NK cell activity and efficacy, focusing on cell source, NK cell activation, and in vivo persistence.

Lymphokine-activated killer cell transplantation after anti-cancer treatment in two aged cats.

Immunotherapy improves both survival and remission rates after cancer surgery in humans, but its veterinary use has been limited. We determined the safety and feasibility of lymphokine-activated killer (LAK) cell transplantation in two aged cats that had undergone surgery for malignancy. Case 1 involved an 18-year-old male Japanese domestic cat. The cat exhibited appetite loss and poor physical activity after the surgical excision of oral squamous cell carcinoma followed by four sessions of radiotherapy, and the owner strongly requested immunotherapy for preventing further deterioration in the animal's quality of life (QOL). We subsequently administered LAK cells three times during a 2-month period. Case 2 involved a 20-year-old female Japanese domestic cat who had undergone mammectomy after a diagnosis of breast adenocarcinoma. The owner strongly requested immunotherapy for QOL maintenance. We administered LAK cells four times over a period of 5 months. Autologous peripheral blood mononuclear cells (PBMCs) fractionated using density gradient centrifugation were cultured in the media containing a high concentration of interleukin-2 and supplemented with 2.5% fetal calf serum. The derived LAK cells were centrifuged, suspended in 10 ml of saline containing 1% of the subject's own blood, and infused into the cephalic vein of the cats over 30 min. The composition ratios of CD3, CD4, CD8, and CD21 were evaluated by flow cytometry. Bacterial culture and endotoxin testing for a sample of LAK cells showed negative results in both the cases. The leukocyte and erythrocyte counts and the body temperature were assessed on days 7, 14, and 21 after the transfusion. No abnormal signs were observed in either case, which confirmed the safety of the procedure. QOL scores showed no significant changes after the treatment, and the body temperature remained steady throughout the treatment. The findings from these cases suggest that the transplantation of LAK cells derived from PBMCs may be safe and feasible for use in cats, regardless of their age.

A Prospective Observational Study of Adoptive Immunotherapy for Cancer Using Zoledronate-Activated Killer (ZAK) Cells - An Analysis for Patients with Incurable Pancreatic Cancer.

BACKGROUND/AIM: Adoptive immunotherapy (AIT) using autologous zoledronate-activated killer (ZAK) cells has been performed for developing a novel modality of cancer treatment. In this study, data series from incurable pancreatic cancer were analyzed. PATIENTS AND METHODS: Patients were treated with AIT using intravenous administration of ZAK cells every 3 to 4 weeks in combination with standard chemotherapy and possible clinical benefits were examined. RESULTS: Seventy-five patients were treated. A median overall survival (OS) time of 6.7 months was achieved for all patients and 13.1 months for those treated 5 times or more, that increased to 14.6 and 18.3 months, respectively, when the previous treatment period of chemotherapy alone was included in the analysis. The disease control rate was 58.5 %. Multivariate regression analysis showed a significant positive correlation between the survival and baseline value of lymphocyte percentage in white blood cell counts (p=0.031). CONCLUSION: The data suggest that AIT using ZAK cells in combination with chemotherapy is safe and feasible and may be effective in prolonging survival for patients with incurable pancreatic cancer. The lymphocyte percentage at baseline may be a good biomarker for predicting the survival benefit of ZAK cell AIT.

Influence of transfusion of lymphokine-activated T killer cells on inflammatory responses in dogs after laparotomy.

The influence of transfusion of lymphokine-activated T killer cells (T-LAK) on inflammatory responses was examined in dogs after laparotomy. Plasma C-reactive protein (CRP) level, cell numbers of peripheral blood lymphocytes (PBLs) and T lymphocyte subsets (CD3(+), CD4(+) and CD8(+)) and mRNA expression levels of cytokines including interleukin (IL)-2, IL-12, IL-4, IL-10 and transforming growth factor (TGF)-ß in peripheral blood mononuclear cells (PBMCs) were measured in dogs with (T-LAK group) or without (control group) a single T-LAK administration immediately after laparotomy. The plasma CRP level initially increased and then decreased to the normal range at 7 days after laparotomy in the T-LAK group, which was earlier than in the control group. The expression level of IL-10 mRNA showed a marked postoperative increase and was significantly higher than the preoperative level on day 7 (P<0.05), whereas the level in the control group showed no clear change after laparotomy. A significant increase in IL-2 mRNA expression level in the T-LAK group was observed on day 14, which was two weeks earlier than in the control group (P<0.05). These results suggest that T-LAK therapy in dogs after laparotomy leads to earlier resolution of postoperative inflammation by production of an anti-inflammatory cytokine (IL-10) in the early phase of the postoperative period and earlier restoration of cell-mediated immunity related to cytokine production by PBMCs.

Stimulation of neuronal cells by culture supernatant of T lymphocytes triggered by anti-CD3 mAb followed by propagation in the presence of interleukin-2.

Performance status (PS) frequently improves occurs in cancer patients who have been infused with their own lymphokine-activated killer T cells (LAK-T). In the present study, a culture supernatant of LAK-T (LAK-T sup) administered to 8-week-old rats caused neurogenesis as evidenced by increased 5-ethynyl-2'-deoxyuridine staining of brain tissues. Intravenous injection of granulocyte-macrophage colony stimulating factor (GM-CSF), a major cytokine in LAK-T sup, had a similar effect. Furthermore, LAK-T sup induced Ca(++) increase in rat hippocampal brain slices that was detected in neuronal cells by emission of Fluo-8 NW at 520 nm. The same effect was observed with an rGM-CSF solution. GM-CSF may activate neuronal cells by stimulating the glial cells that surround and attach to them. If so, GM-CSF and LAK-T sup may improve the motor neurons of patients with amyotrophic lateral sclerosis. The neurogenerative effect of GM-CSF in LAK-T sup may also help improve brain function in aged adults including those with dementia such as Alzheimer's disease.

Activated and expanded natural killer cells target osteosarcoma tumor initiating cells in an NKG2D-NKG2DL dependent manner.

Current therapies fail to cure most metastatic or recurrent bone cancer. We explored the efficacy and the pathways involved in natural killer (NK) cells' elimination of osteosarcoma (OS) cells, including tumor initiating cells (TICs), which are responsible for chemotherapy resistance, recurrence, and metastasis. The expression of ligands for NK cell receptors was studied in primary OS cell lines by flow cytometry. In vitro cytotoxicity of activated and expanded NK (NKAE) cells against OS was tested, and the pathways involved explored by using specific antibody blockade. NKAE cells' ability to target OS TICs was analyzed by flow cytometry and sphere formation assays. Spironolactone (SPIR) was tested for its ability to increase OS cells' susceptibility to NK cell lysis in vitro and in vivo. We found OS cells were susceptible to NKAE cells' lysis both in vivo and in vitro, and this cytolytic activity relied on interaction between NKG2D receptor and NKG2D ligands (NKG2DL). SPIR increased OS cells' susceptibility to lysis by NKAE cells, and could shrink the OS TICs. Our results show NKAE cells target OS cells including the TICs compartment, supporting the use of NK-cell based immunotherapies for OS.

T-cell-associated cellular immunotherapy for lung cancer.

PURPOSE: The aim of the present study was to discuss recent findings on the role of T cells in lung cancer to provide information on their potential application, especially in cellular immunotherapy. METHODS: Data on the different types of T cells that are currently used for the treatment of lung cancer were obtained by searching the PUBMED database. RESULTS: Cytotoxic T lymphocytes, natural killer T cells, γδ T cells, lymphokine-activated killer cells, tumor-infiltrating lymphocytes, cytokine-induced killer cells and gene-modified T cells were analyzed to determine the benefits and drawbacks of their application in the treatment of lung cancer. Advances in the study of their antitumor mechanisms and directions for future research were discussed. CONCLUSIONS: T cells are critical for tumorigenesis and therefore important targets for the treatment of lung cancer. T-cell-associated cellular immunotherapy opens up a window of opportunity for the development of complementary methods to traditional lung cancer treatments, which warrants further investigation to improve the clinical outcomes of lung cancer patients.

CD16+CD56+ cells are a potential culprit for hematuria in IgA nephropathy.

BACKGROUND: Hematuria is the first manifestation of urinary abnormality in immunoglobulin A nephropathy (IgAN). Hematuria has recently been reported as a risk factor for deterioration of renal function; however, its cause remains unknown. METHODS: We analyzed the surface marker of peripheral blood mononuclear cells before and immediately after tonsillectomy in IgAN patients and controls (chronic tonsillitis or tonsillar hypertrophy) by flow cytometry and investigated the association with hematuria. To prove our hypothesis that NK cells induce hematuria, we administered IL-12, activator of NK cells, to HIGA mice. In addition, we transferred cultured NK cells to nude rats and transferred the CD16(+)CD56(+) cells, including NK cells, that are derived from the peripheral blood of IgAN patients immediately after tonsillectomy to nude rats to assess the hematuria level and renal histology of the recipients. We also performed cytotoxicity assays against glomerular endothelial cells by NK cells. RESULTS: We found that IgAN patients who showed rapid deterioration of hematuria after tonsillectomy also displayed a significant increase in CD16(+)CD56(+) cells in the peripheral blood immediately after tonsillectomy. Exogenous administration of IL-12 to HIGA mice induced hematuria. Adoptive transfer of either cells of an NK cell line, or of CD16(+)CD56(+) cells derived from IgAN patients, into nude rats induced hematuria in the recipients. In vitro analysis showed that NK cells exert cytotoxic activity toward human glomerular endothelial cells in a dose-dependent manner. CONCLUSIONS: CD16(+)CD56(+) cells seem to be responsible for hematuria in IgAN.

Targeted Hsp70 expression combined with CIK-activated immune reconstruction synergistically exerts antitumor efficacy in patient-derived hepatocellular carcinoma xenograft mouse models.

The patient-derived tumor xenograft (PDTX) models can reproduce a similar natural genetic background and similar biological behaviors to tumor cells in patients, which is conducive to the assessment of personalized cancer treatment. In this study, to verify the targeting and effectiveness of the therapeutic strategy using a Survivin promoter-regulated oncolytic adenovirus expressing Hsp70, the PDTX models of hepatocellular carcinoma (HCC) were established in nude mice and the cytokine-induced killer (CIK) cells were intravenously infused into mice to partially reconstruct the mouse immune function. The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited. However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells. Oncolytic adenovirus mediated the specific expression of Hsp70 in cancer tissues allowed the CIK chemotaxis, and induce the infiltration of CD3+ T cells in tumor stroma, thereby exhibiting anti-tumor activity. The anti-tumor effect was more effective for the highly malignant tumor xenografts with highly Survivin expression. This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts.

Irradiated mononuclear cells express significant in vitro cytotoxic activity: promise for in vivo clinical efficacy of irradiated mismatched donor lymphocytes infusion.

BACKGROUND: Relapse of the original disease remains the most common cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-SCT). Patients who relapse post-allo-SCT can achieve prolonged remission after donor lymphocyte infusion. Donor lymphocyte infusion as well as other immunotherapeutic strategies are usually complicated by severe graft versus host disease. AIM: In the present study, we examined the effect of irradiation on the cytotoxic activity of mononuclear cells (MNCs). MATERIALS & METHODS: Cytotoxic activity of fresh and irradiated MNCs from healthy donors was tested against the leukemic cell line K562 and against fresh leukemic cells from patients with acute myeloid leukemia. Cytotoxicity was assessed by using a flow-cytometry assay. RESULTS & DISCUSSION: Interestingly, we observed that 25 Gy irradiated MNCs retain significant cytotoxic activity against K562. Based on these in vitro data, the safety and efficacy of irradiated haploidentical, IL-2-activated lymphocytes were tested in six patients after allo-SCT. Acute skin graft versus host disease developed in two patients and was resolved after a short course of steroids. One patient with mixed chimera converted to full donor chimera after infusion of irradiated donor cells. CONCLUSION: The efficacy of irradiated haploidentical lymphocytes should be further tested in a larger number of patients.

Cytokine Induced Killer (CIK) cells for the treatment of haematological neoplasms.

Cytokine Induced Killer (CIK) cells are in vitro activated human CD8 T cells which have maintained several characteristics of T-EMRA cells and additionally acquired non specific anti tumoral cytotoxicity and CD56 overexpression, thus representing a cell population with double T and NK phenotype. Due to their in vivo intratumoral homing and lack of Graft versus Host (GVH) reactivity, CIK cells have been extensively used in cancer patients either in autologous or allogeneic contexts. Here we summarise CIK main biological features as well as their most prominent clinical results.

Cytokine therapy.

Cytokines are a heterogeneous group of soluble small polypeptides or glycoproteins, which exert pleiotropic and redundant effects that promote growth, differentiation and activation of normal cells. Cytokines can have either pro- or anti-inflammatory activity and immunosuppressive activity, depending on the microenvironments. The tumor microenvironment consists of a variable combination of tumor cells, endothelial cells and infiltrating leukocytes, such as macrophages, T-lymphocytes, natural killer (NK) cells, B cells and antigen-presenting cells (APCs). Cytokine production acts as a means of communication in the tumor microenvironment. In this article, we review the cross-talk between cytokines in the tumor environment and the cytokine therapies that have been used till date for glioma treatment.

Adoptive cell transfer therapy for malignant gliomas.

To date, various adoptive immunotherapies have been attempted for treatment of malignant gliomas using nonspecific and/or specific effector cells. Since the late 1980s, with the development of rIL-2, the efficacy of lymphokine-activated killer (LAK) cell therapy with or without rIL-2 for malignant gliomas had been tested with some modifications in therapeutic protocols. With advancements in technology, ex vivo expanded tumor specific cytotoxic T-lymphocytes (CTL) or those lineages were used in clinical trials with higher tumor response rates. In addition, combinations of those adoptive cell transfer using LAK cells, CTLs or natural killer (NK) cells with autologous tumor vaccine (ATV) therapy were attempted. Also, a strategy of high-dose (or lymphodepleting) chemotherapy followed by adoptive cell transfer has been drawing attentions recently. The most important role of these clinical studies using cell therapy was to prove that these ex vivo expanded effector cells could kill tumor cells in vivo. Although recent clinical results could demonstrate radiologic tumor shrinkage in a number of cases, cell transfer therapy alone has been utilized less frequently, because of the high cost of ex vivo cell expansion, the short duration of antitumor activity in vivo, and the recent shift of interest to vaccine immunotherapy. Nevertheless, NK cell therapy using specific feeder cells or allergenic NK cell lines have potentials to be a good choice of treatment because of easy ex vivo expansion and their efficacy especially when combined with vaccine therapy as they are complementary to each other. Also, further studies are expected to clarify the efficacy of the high-dose chemotherapy followed by a large scale cell transfer therapy as a new therapeutic strategy for malignant gliomas.

Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma.

OBJECTIVES: In the current study, we have evaluated the clinical and immunological responses in patients with advanced pancreatic carcinoma who received dendritic cell (DC)-based immunotherapy in combination with gemcitabine and/or S-1. METHODS: Dendritic cell-based immunotherapy (DC vaccine alone or DC vaccine plus lymphokine-activated killer [LAK] cell therapy) in combination with gemcitabine and/or S-1 has been carried out in 49 patients with inoperable pancreatic carcinoma refractory to standard treatment. RESULTS: Of 49 patients, 2 patients had complete remission, 5 had partial remission, and 10 had stable disease. Prolongation of survival in this cohort was highly likely (median survival, 360 days). Survival of patients receiving DC vaccine and chemotherapy plus LAK cell therapy was longer than those receiving DC vaccine in combination with chemotherapy but no LAK cells. Increased numbers of cancer antigen-specific cytotoxic T cells and decreased regulatory T cells were observed in several patients on immunotherapy, but increased overall survival time tended to be associated only with the latter. None of the patients experienced grade 3 or worse adverse events during the treatment period. CONCLUSIONS: Dendritic cell vaccine-based immunotherapy combined with chemotherapy was shown to be safe and possibly effective in patients with advanced pancreatic cancer refractory to standard treatment.

[Examination of the patient serum presenting a CEA false high value after cancer immuno-cell therapy].

False positive elevation of carcinoembryonic antigen (CEA) was observed in a 65-year-old woman who was treated with dendritic cell therapy (DCT) and activated lymphocyte therapy (ALT) for intrahepatic cholangiocarcinoma. Three months after the initiation of these therapies, her CEA value measured by AIA (TOSOH) began to increase without any evidence of worsening of cholangiocarcinoma. CEA was measured by several different methods, and only the result measured by AIA was high, indicating the presence of a false positive phenomenon. To clarify this phenomenon, we evaluated the patient's serum precisely. Gel filtration chromatography of her serum showed that CEA was detected in the elution fraction of IgG, which was different from the reference samples. Furthermore, this peak disappeared after incubation of patient's CEA and HBR-1. The immunoglobulin absorption test revealed that CEA value was decreased only after absorption of IgG and absorption tests using HBR-1 and MAK-absorbents showed a dramatic decrease in CEA value. These findings indicated the presence of IgG type human anti-mouse antibodies (HAMA), which interfered the measurement by AIA. Although we could not identify the reason why HAMA was produced in this patient, the facts that the false positive phenomenon was observed after the initiation of DCT and ALT, and that CEA value decreased after theses therapies were discontinued, indicated that immuno-modulaton by DCT and ALT may have a close relationship to HAMA production. It was probable that DCT and ALT activated preexisting heterophile-antibody-producing cells, which stimulated HAMA production. The incidence of such false positive reaction of CEA by HAMA in patients with DCT and ALT was low, but as the number of the patient with immuno-cell therapy increases, the incidence of such phenomenon surely increases. Because HAMA reacts to all types of immunoassay, careful attention should be paid to the evaluation of laboratory findings in patients undergoing with such immuno-cell therapies.

LAK cell therapy of AML: not to be lost in translation.

Expanding highly cytotoxic lymphokine activated natural killer cells and cytokine induced killer T cells by Good Manufacturing Practice-quality cultures is an important step toward effective use of those killer cells to treat acute myeloid leukemia patients receiving stem cell transplantation. Elucidating how those killer cells recognize leukemia stem cells will be the next challenge.

Bone marrow mesenchymal stem cells reduce the antitumor activity of cytokine-induced killer/natural killer cells in K562 NOD/SCID mice.

Adoptive cellular immunotherapy is an important treatment to eliminate residual tumor cells after hematopoietic stem-cell transplantation. Bone marrow mesenchymal stem cells (MSC) have previously been shown to exert immunoregulation functions, including inhibition of proliferation and killing activities of T cells and natural killer (NK) cells in vitro and reduction of the graft-versus-host disease. MSC can survive in vivo for a long period of time, the influence of MSC on the antitumor activity of subsequently infused immune killer cells is not clear. The aim of this study was to investigate the influences of MSC infused via different paths and at different times on the antitumor activities of cytokine-induced killer (CIK)/NK cells derived from umbilical cord blood in K562 NOD/SCID mice. The potential interaction mechanisms of MSC and CIK/NK cells infused through different paths using different intervals in vivo were subsequently explored. The results show that the antitumor activities of CIK/NK cells was inhibited by MSC when injected via the same path (tail vein), and the suppressive effect of MSC on CIK/NK cells were less pronounced when they were injected separately through different paths. There were no effects of MSC on the antitumor activities of CIK/NK cells if the MSC and CIK/NK cells were injected with a 48-h interval. Moreover, the suppressive effect continuous, even if MSC were infused 48 h earlier than CIK/NK cells. It suggests that pre-injected MSC can reduce the antitumor activities of CIK/NK cells in vivo. The probable mechanisms are that MSC and CIK/NK cells might have a greater opportunity to meet and interact if they are injected simultaneously via the same path. The suppression of MSC on CIK/NK cells in vivo mainly takes place in the reticuloendothelial system, including the lung and the liver.

Decrease of the regulatory T-cell population by adoptive T-cell transfer in a mouse colorectal cancer transplant model.

We examined the effects of adoptive T-cell transfer (ACT) on the population of regulatory T cells (Tregs) in a mouse colorectal cancer transplant model. In an in vivo study, Treg populations in Balb/c mice colon26 transplant model after ACT were analyzed in peripheral blood, local lymph node, and tumor. In an in vitro study CD4+ cells were cultured in medium containing TGF-beta to induce Tregs. LAK cells were added or not in this Treg induction system. Treg induction after coculture with LAK was investigated. We also studied the role of IFN-gamma in the mechanism of Treg induction. Tregs in the draining lymph nodes and tumor were significantly suppressed by ACT. The induction of Tregs in vitro was inhibited by coculture with LAK cells. Furthermore, Tregs in the cultured cells were significantly inhibited by addition of exogenous IFN-gamma. Moreover, Tregs were increased by addition of IFN-gamma mAb. ACT may decrease Tregs in tumor-bearing hosts. One of the mechanisms is considered to be IFN-gamma inhibiting the induction of Tregs.