Cell-mediated Immunotherapy Research Articles

Inhibition of phosphoantigen‐mediated γδ T‐cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.

Natural killer T cell-mediated immunotherapy for malignant diseases

Human invariant Valpha24 Natural Killer T (NKT) cells are unique lymphocyte subsets, characterized by an invariant T-cell receptor Valpha24 chain paired with Vbeta11. Recent findings have highlighted the role of NKT cells in tumor immunity. Valpha24 NKT cells are activated by a specific glycolipid ligand, alpha-Galactosylceramide and rapidly produce high levels of cytokines upon stimulation, thereby modulating other immune cells such as NK cells antigen-specific CD4+ and CD8+ T cells and dendritic cells. Abnormalities in the numbers and functions of Valpha24 NKT cells have been observed in patients with various malignant diseases. Therefore, therapeutic strategies have recently focused on the reconstitution of an adequate number of functionally sufficient Valpha24 NKT cells which is thought to be logical and reasonable for cancer treatment. The quantitative alteration and functional impairment of circulating Valpha24 NKT cells are herein reviewed in various cancer-bearing patients and the progress to date in the clinical applications of NKT cell-based tumor immunotherapy is summarized.

Immunotherapy of Head and Neck Cancer: Current and Future Considerations

Patients with head and neck squamous cell carcinoma (HNSCC) are at considerable risk for death, with 5-year relative survival rates of approximately 60%. The profound multifaceted deficiencies in cell-mediated immunity that persist in most patients after treatment may be related to the high rates of treatment failure and second primary malignancies. Radiotherapy and chemoradiotherapy commonly have severe acute and long-term side effects on immune responses. The development of immunotherapies reflects growing awareness that certain immune system deficiencies specific to HNSCC and some other cancers may contribute to the poor long-term outcomes. Systemic cell-mediated immunotherapy is intended to activate the entire immune system and mount a systemic and/or locoregional antitumor response. The delivery of cytokines, either by single cytokines, for example, interleukin-2, interleukin-12, interferon-γ, interferon-α, or by a biologic mix of multiple cytokines, such as IRX-2, may result in tumor rejection and durable immune responses. Targeted immunotherapy makes use of monoclonal antibodies or vaccines. All immunotherapies for HNSCC except cetuximab remain investigational, but a number of agents whose efficacy and tolerability are promising have entered phase 2 or phase 3 development.

Natural killer T cell-mediated immunotherapy for malignant diseases

Ischemia/reperfusion (I/R) injury remains a major problem in solid organ transplantation, as it adversely impacts both short and long term outcomes. It has been well established that the innate immune system plays a significant role in the pathogenesis of I/R injury. In contrast, the proximal molecular signaling events that initiate activation of the innate immune system are less clear. Recent findings have demonstrated that Toll-like receptors (TLR) play a role in I/R injury. Specifically, TLR4 is central to early activation of the innate immune response in the setting of I/R. Furthermore, recent evidence has shown that endogenous molecules, such as high mobility group box-1 (HMGB1) and others that are released from ischemic, damaged, or dying cells and tissues in the setting I/R, can serve as triggers for activation of the innate immune system and exacerbate tissue injury. This evolving body of literature, which has provided insight into the early molecular events that activate the innate system after I/R, is reviewed here.

Successful cell‐mediated cytokine‐activated immunotherapy for relapsed acute myeloid leukemia after hematopoietic stem cell transplantation

Acute myeloid leukemia (AML) is an extremely aggressive disease with a high relapse rate even after allogeneic hematopoietic stem cell transplantation (HSCT). We report the successful outcome of cell-mediated cytokine-activated immunotherapy in a high-risk pediatric AML patient who relapsed shortly after allogeneic HSCT. Donor lymphocyte infusion along with interferon induced a graft-versus-leukemia effect, presenting as a reversible episode of graft-versus-host disease, which led to stable complete donor chimerism and total eradication of AML for over 24 months, at the time of this report. The curative potential of immunotherapy in hematological malignancies is discussed.

Natural killer cell‐mediated lysis of freshly isolated human tumor cells

Studies on interactions between natural killer (NK) cells and freshly isolated human tumor cells are still relatively scarce. With respect to the understanding of NK cell interactions with human tumors in vivo, and attempts to better predict the outcome of NK cell-mediated adoptive immunotherapy, such studies merit further attention. They may provide information that is not readily evident through studies of human tumor cell lines established through long-term culturing in vitro. The latter undergo several changes and adaptations to their environment, many of which may affect interactions with NK cells. Studies of NK cell interactions with freshly explanted human tumors, however, are cumbersome for several reasons including; often poor accessibility of human tumor material per se, difficulties in sample processing and contamination of tumors with nontumor cells, spontaneous apoptosis of tumor cells, as well as methodological challenges in assessing tumor cell lysis following interactions with NK cells. Here, we review the current knowledge on NK cell interaction with freshly isolated human tumors.

Cognitive Study of Reactivity to IPH1101 of Peripheral γδ T Lymphocytes from Chronic Myeloid Leukemia, Multiple Myeloma and Follicular Lymphoma Patients.

Background: IPH1101, a chemically-synthesized, structural analogue of γδ T lymphocyte natural phosphoantigens, combined with low doses of IL-2 induces a highly selective proliferation of γδ T cells, non conventional lymphocytes bearing potent effector and regulatory immune functions. Today, IPH1101 is tested in Phase II clinical trials in different indications and settings (alone or in combination), as the first specific γδ T cell-mediated immunotherapy. Whereas γδT cells from healthy donors always proliferate in response to IPH1101 + IL-2, cells from cancer patients often present moderate to very strong impaired proliferative capacity. The reasons for this defect are still unclear, but might result from a suppressive effect induced by the tumour itself. Thus, defective γδ T cell responses might be partly disease-dependent. In some cases, the decreased ability to respond to IPH1101 might also correlate with the stage of the disease or the nature of previous or ongoing treatments. In order to explore in which cancer indications or clinical settings γδ T cell pharmacology is impaired or fully maintained, we have set up a quantitative standardized in vitro “IPH1101 sensitivity test” that requires only a small sample of patient's PBMC. The objective of this ongoing ex-vivo observational study is to identify types of cancers and settings for which γδ T cell immunotherapy using IPH1101 treatment may be beneficial. Here are reported results from 3 hematological indications: multiple myeloma (MM), follicular lymphoma (FL) and chronic myeloid leukaemia (CML) receiving long term imatinib treatment.Method: Patients with MM, FL and CML have been enrolled at 2 French sites. A small sample of blood (20 mL) is sufficient to prepare PBMCs and culture them in the presence of IPH1101 and IL-2. Results on the extent of in vitro amplification of cells by IPH1101 are available within 8 days and are expressed as% of γδ T cells in the culture andtotal amplification rate of γδ T cells.Results: Nineteen patients with MM, 31 with FL and 19 with CML receiving imatinib were evaluable in this study. One patient by indication was found strictly not sensitive to IPH1101 stimulation ex vivo, meaning that their γδT cells showed no signs of proliferation in culture. Samples from patients with MM and, to a lesser extent, FL showed a clear impairment of their proliferative response as compared to samples from healthy individuals, with 32% and 10% of MM and FL samples, respectively, demonstrating an impaired response. Finally, in imatinib-treated CML patients, 94% PBMC samples had γδ T cells with full proliferative capacity in response to IPH1101.Conclusion: Ex vivo, γδ T cells from multiple myeloma and follicular lymphoma patients show a low proportion of moderate to strong impairment of their proliferative capacity in response to their specific stimulus IPH1101. For future γδ T cell based immunotherapy trials in these indications, it might be beneficial to consider selecting patients based on such a criteria. In CML patients undergoing long-term treatment with imatinib, γδ T cells have maintained full ability to respond to their specific stimulus (despite the well-known immunosuppressive effects of imatinib). Thus, these results confirm the biological feasibility of combining imatinib and IPH1101 in a clinical trial in CML.

Generation of Antitumor Invariant Natural Killer T Cell Lines in Multiple Myeloma and Promotion of Their Functions via Lenalidomide: A Strategy for Immunotherapy

CD1d-restricted invariant natural killer T (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma hamper their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in multiple myeloma. We activated and expanded iNKT cells from multiple myeloma patients with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells, characterized their antitumor effects by the cytokine production profile and cytotoxicity against multiple myeloma cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary multiple myeloma cells and its function to activate iNKT cells. We established highly purified functional iNKT cell lines from newly diagnosed and advanced multiple myeloma patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to alpha-GalCer-pulsed primary multiple myeloma cells, CD1d-transfected MM1S cell line, and dendritic cells. Moreover, iNKT cell lines displayed strong cytotoxicity against alpha-GalCer-pulsed primary multiple myeloma cells. Importantly, lenalidomide further augmented the Th1 polarization by iNKT cell lines via increased Th1 cytokine production and reduced Th2 cytokine production. We also showed that CD1d was expressed in primary multiple myeloma cells at mRNA and protein levels from the majority of multiple myeloma patients, but not in normal plasma cells and multiple myeloma cell lines, and CD1d(+) primary multiple myeloma cells presented antigens to activate iNKT cell lines. Taken together, our results provide the preclinical evidence for the iNKT cell-mediated immunotherapy and a rationale for their use in combination with lenalidomide in multiple myeloma treatment.

How tumours escape mass destruction

It is now well established that the immune system can control neoplastic development and growth in a process termed immunosurveillance. A link between host immunosurveillance and neoplastic progression is revealed in cases where the immune response becomes compromised due to genetic or other pathological conditions, resulting in a substantially increased incidence and rate of spontaneous tumour formation in both preclinical animal models and patients. It has also been demonstrated in tumour-bearing hosts that the tumorigenic process itself can promote a state of immunosuppression that, in turn, facilitates neoplastic progression. The ability of neoplastic populations to induce a hostile microenvironment through both cell contact-dependent and -independent immunosuppressive networks is a significant barrier to effective cell-mediated immunity and immunotherapy. Thus, a competent immune system is integral for the control of neoplastic disease, and dissecting the plethora of tumour escape mechanisms that disrupt this essential host defense capability is integral for the development of effective immunotherapeutic paradigms.

Cell fusion: from hybridoma to dendritic cell-based vaccine

The deployment of dendritic cell (DC) and tumor cell fusions is increasing in tumor immunotherapy. In animal and human studies, fusion cell vaccines have been shown to possess the elements essential for processing and presenting tumor antigens to host immune cells, for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens. Moreover, fusion cell vaccines provide protection against challenge with tumor cells and mediate regression of established tumors. Despite these unique features of fusion cell vaccines and the observation of tumor eradication in animal studies, limited success has occurred in clinical trials. This article reviews the methods used for optimizing the preparation and selection of DC–tumor fusion cells and analyzes factors influencing the success or failure of fusion cell-mediated immunotherapy. In addition, we discuss the challenges facing effective fusion cell vaccine production, including factors in preparation, selection and quality control of fusion cell vaccines, as well as approaches for enhancing anti-tumor immunity.

Toll-like receptor agonists and invariant natural killer T-cells enhance antibody-dependent cell-mediated cytotoxicity (ADCC)

huHMFG-1 (AS1402) is a humanised IgG1 against MUC1, which exerts tumour cell killing through antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Here, we explored the capacity of invariant NKT (iNKT) cells, which are known to activate NK cells, and toll-like receptor (TLR) ligands which activate both iNKT and NK cells, to enhance huHMFG-1-ADCC. Addition of iNKT cells, as well as TLR2/6, 7, 8 and 9 agonists to PBMC improved the efficacy of huHMFG-1. These results suggest that transfer of ex vivo expanded iNKT cells or TLR agonist treatment may improve the efficacy of NK cell-mediated antibody-based tumour immunotherapies.

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways

Introduction Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated. Methods and results All EWS cell lines tested ( n = 7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis ( n = 11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro. Conclusion These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.

Potentiation of the immunotherapeutic effect of autologous dendritic cells by pretreating hepatocellular carcinoma with low-dose radiation

To determine whether exposing hepatocellular carcinoma (HCC) to low dose radiation increases the efficacy of dendritic cell-mediated immunotherapy for HCC. Tumour specimens collected from 20 recruited patients with HCC were cultured in primary culture (half successfully) and then exposed to low-dose radiation (0.5 Gy). Immature DCs derived from peripheral blood monocytes of patients were pulsed with autologous HCC cell lysates and matured with a cytokine cocktail. Autologous tumour lysate-pulsed DCs (TLP-DCs) were used to stimulate mixed lymphocytes, which were then tested for inhibitory effect on the growth of HCC cells. Surface markers of immunogenicity on primary HCC cells, MHC, and Fas were investigated before and after low-dose irradiation. Exposing HCC cells to low-dose (0.5 Gy) radiation enhanced the immunotherapeutic effect of TLP-DC-stimulated lymphocytes. Growth inhibition increased from 50.6+/-7.5% without irradiation to 74.3+/-4.3% with radiation. The expression of MHC class ll and Fas was upregulated after irradiating HCC cells. Exposing tumour cells to a low dose of radiation can enhance the immunotherapeutic effect of the autologous tumor lysate-pulsed DC vaccine.

DNA vaccines for cervical cancer: from bench to bedside

More than 99% of cervical cancers have been associated with human papillomaviruses (HPVs), particularly HPV type 16. The clear association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. Although the recently licensed preventive HPV vaccine, Gardasil, has been shown to be safe and capable of generating significant protection against specific HPV types, it does not have therapeutic effect against established HPV infections and HPV-associated lesions. Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. It has been established that T cell-mediated immunity is one of the most crucial components to defend against HPV infections and HPV-associated lesions. Therefore, effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. DNA vaccines have emerged as an attractive approach for antigen-specific T cell-mediated immunotherapy to combat cancers. Intradermal administration of DNA vaccines via a gene gun represents an efficient way to deliver DNA vaccines into professional antigen-presenting cells in vivo. Professional antigen-presenting cells, such as dendritic cells, are the most effective cells for priming antigen-specific T cells. Using the gene gun delivery system, we tested several DNA vaccines that employ intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded model antigen HPV-16 E7. Furthermore, we have developed a strategy to prolong the life of DCs to enhance DNA vaccine potency. More recently, we have developed a strategy to generate antigen-specific CD4(+) T cell immune responses to further enhance DNA vaccine potency. The impressive pre- clinical data generated from our studies have led to several HPV DNA vaccine clinical trials.

Activated T Cell-Mediated Immunotherapy with a Chimeric Receptor Against CD38 in B-Cell Malignancies.

CD38 is expected to be one of the most useful molecular targets on the surface of malignant B-cells. T cell-mediated immunotherapy with a chimeric receptor could provide a powerful tool for treating cancer. However, since CD38 is also expressed on effector cells such as activated T cells, NK cells, and monocytes, these cells with the chimeric receptor could be eliminated by autologous cytotoxicity through the interaction with the antigen. In this study, we developed a novel methodology for enhancing the survival and clonal expansion of T lymphocytes expressing an anti-CD38 chimeric receptor. Hut78 T cells, which express very little CD38, retrovirally transduced with the anti-CD38 chimeric receptor showed powerful cytotoxic activity against B cell lines expressing CD38, such as HT (lymphoma), RPMI8226 (myeloma), 380 (ALL-Ph1−) and OP-1 (ALL-Ph1+) cells (mean specific cytotoxicity was 97.94% ± 0.31% after four days of culture in vitro). However, in activated human T cells and Jurkat cells constitutively expressing CD38, the recovery rate of cells transduced with the chimeric receptor was extremely low, because the cells eradicated each other and/or themselves by inducing apoptosis. To block the interaction of the anti-CD38 chimeric receptor with CD38 antigen, we incubated activated T cells and Jurkat cells in medium supplemented with an anti-CD38 antibody before the transduction. The number of viable cells harvested after the transduction was dramatically increased by the antibody in a dose-dependent manner. Using this method, we prepared human peripheral T cells bearing the chimeric receptor and injected them into NOD/SCID mice, which were transplanted with HT cells labeled with luciferase. Lucuferase activity was not detectable in 13 days in five of six mice with T cells transduced with the chimeric receptor. In contrast, the activity had a rapid and steady increase in all of the mice injected with vector-transduced T cells. These results clearly showed that even though human peripheral T cells express any molecule on their surface, an antibody could protect T cells transduced with a chimeric receptor-containing vector from cytolysis, and apoptosis. These findings may provide us with a powerful tool for improving T cell-mediated targeting therapy.

Anti-Tumor Cytotoxicity of γδ T Cells Expanded from Blood Cells of Myeloma and Leukemia Patients Against Self Tumor Cells — Enhancement of the Anti-Tumor Cytotoxicity by Type I IFN, Dendritic Cells, and Activated αβ T Cells.

In order to establish an efficient gd T cell-mediated immunotherapy for hematological malignancies, we tried to clarify whether γδ T cells could be expanded from blood cells of patients with myeloma, lymphoma and acute leukemia by culture with zoledronate and a low dose of IL-2 and whether the expanded patients' γδ T cells could kill tumor cells including self tumor cells with sparing normal clone cells. In addition, we explored the methods to enhance the anti-tumor cytotoxicity of the expanded γδ T cells by activating them with type I IFN, monocyte-derived dendritic cells (mo-DCs), or ab T cells. Although γδ T cells could be expanded in patients with myeloma, lymphoma and leukemia as well as normal persons, the amplification rates of gd T cells before and after the culture were varied from patient to patient in the patients with hematological malignancies. γδ T cells generated in patients with myeloma and lymphoma showed a potent cytotoxic ability against myeloma/lymphoma cell lines (RPMI8226, Daudi) as shown in γδ T cells generated in normal persons. In addition, γδ T cells generated in a patient with myeloma and acute leukemia showed a cytotoxic ability against self myeloma or leukemia cells freshly prepared from bone marrow. However, the same γδ T cells were not cytotoxic to normal lymphocytes of the patients. Then the expanded γδ T cells were stimulated with type I IFN, mo-DCs, or αβ T cells and the activation (CD69 expression) and cytotoxicity against tumor cells were examined. By the stimulation with type I IFN, the expression of CD69 and Trail of γδ T cells was increased and the cytotoxic ability of γδ T cells was enhanced at dose-dependent manner of type I IFN. CD69 expression on γδ T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after the addition of mo-DCs of either autologous or allogeneic origin. Activation of γδ T cells with mo-DCs enhanced anti-tumor cytotoxicity of γδ T cells against RPMI8226 and CML blastic crisis cell line (C2F8) in an effector-to-target ratio-dependent manner. Although CD69 expression of γδ T cells was enhanced by the co-culture with allogeneic ab T cells, autologous ab T cells couldn't activate γδ T cells. However, autologous ab T cells stimulated with IL-2 or PHA could induce the activation of γδ T cells. The activation of γδ T cells with stimulated αβ T cells required cell-to-cell interaction. These findings suggested that αβ T cells stimulated by allogeneic γδ T cells could activate the same allogeneic γδ T cells. The present data demonstrated that γδ T cells, which could be expanded in vitro from blood cells of the patients with myeloma, lymphoma and leukemia by culture with zoledronate and IL-2, possess an enough cytotoxic ability against tumor cells including self tumor cells with sparing normal cells. These findings suggested that in vitro generated patients' γδ T cells could be applied to γδ T cell-mediated immunotherapy for hematological malignancies. Besides, potent γδ T cells activated by type I IFN, mo-DCs or activated αβ T cells were considered to be applicable for γδ T cell-mediated immunotherapy.

Anti-tumor cytotoxicity of γδ T cells expanded from peripheral blood cells of patients with myeloma and lymphoma

In order to establish an efficient gammadelta T cell-mediated immunotherapy for hematological malignancies, we attempted to evaluate cytotoxicity against tumor cells by gammadelta T cells, which were generated from blood cells of patients with myeloma and lymphoma by culturing with zoledronate and a low dose of IL-2. Although gammadelta T cells were expanded in patients with myeloma and lymphoma as well as normal persons, the amplification rates of gammadelta T cells before and after culturing varied from patient to patient in myeloma and lymphoma. gammadelta T cells generated in patients with myeloma and lymphoma showed a potent cytotoxic ability against myeloma/lymphoma cell lines as shown in gammadelta T cells generated in normal subjects. In addition, gammadelta T cells generated in a patient with myeloma showed a cytotoxic ability against self myeloma cells freshly prepared from bone marrow. However, the same gammadelta T cells were demonstrated to be non-cytotoxic to normal cells of the patient. These data demonstrated that gammadelta T cells, which could be expanded in vitro from blood cells of patients with myeloma and lymphoma by culturing with zoledronate and IL-2, possess a sufficient cytotoxic ability against tumor cells. These findings suggested that in vitro generated patients' gammadelta T cells could be applied to gammadelta T cell-mediated immunotherapy for hematological malignancies.

Enhancement of Anti-tumor Cytotoxicity of Expanded .GAMMA..DELTA. T Cells by Stimulation with Monocyte-derived Dendritic Cells

In order to establish the method of generating powerful gammadelta T cells for anti-tumor immunotherapy, we investigated the effects of monocyte-derived dendritic cells (mo-DCs) on anti-tumor cytotoxicity of expanded gammadelta T cells. Activation of gammadelta T cells co-cultured for 2-3 days with immature or mature mo-DCs was evaluated by CD69 expression and anti-tumor cytotoxicity using two assays : the 5- (and 6-) carboxyfluorescein diacetate, succinimidyl ester-based cytotoxicity assay and the calcein-AM-based Terascan assay. gammadelta T cells were used as effector cells and myeloma cell line (RPMI8226) or chronic myelogenous leukemia blastic crisis cell line (C2F8) were used as target cells. CD69 expression on gammadelta T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after addition of mo-DCs of either autologous or allogeneic origin. Activation of gammadelta T cells with mo-DCs enhanced anti-tumor cytotoxicity of gammadelta T cells against RPMI8226 and C2F8 in an effector-to-target ratio-dependent manner. Activation of gammadelta T cells by mo-DCs was associated with the enhancement of anti-tumor cytotoxicity of gammadelta T cells. Potent gammadelta T cells activated by mo-DCs were considered to be applicable to an efficient gammadelta T cell-mediated immunotherapy for tumors.

NKT cell-mediated immunotherapy for hematological malignancies

NKT cell-mediated immunotherapy for hematological malignancies

Rapid and Massive Expansion of Cord Blood Derived Cytokine Induced Killer (CIK) Cells: An Innovative Proposal for the Treatment of Leukemia Relapse after Cord Blood Transplantation.

Cytokine induced killer cells (CIK) are CD3+/CD56+ T/NK cells with cytotoxic potential against leukemic and other tumor cells but not normal bone marrow in vitro and in vivo. They are expanded in vitro with rhIL-2 after stimulation of peripheral blood mononuclear cells with OKT3 and IFN-γ. We have shown in a recent phase I study that 107/kg allogeneic CIK cells can be safely given to patients relapsing after allogeneic bone marrow transplantation and show evidence of anti-leukemic activity in vivo with very little GVHD. Cord blood (CB) transplantation is progressively becoming an extensively used treatment for patients with malignant disorders. One major limitation of this procedure is the lack of donor derived cells to perform donor lymphocyte infusions in case of relapse. In order to be able therefore to extend the use of CIK cells to the CB transplantation setting, we have standardised a 21 days expansion protocol to produce CIK cells starting from very small amounts of nucleated cells isolated from cord blood. Using this protocol, 15x106 mononuclear cells (MNC) from CB containing a mean 0.3x106 CD3+/CD56+ yielded on average 805 x 106 MNC (50 fold expansion) containing 630 x106 CD3+/CD56+ cells (corresponding to a fold expansion of 1860 for CIK). In order to transfer the method to a clinical setting, we explored the possibility of expanding the residual cells recovered from the empty bags after CB transplantion. Three used CB bags were returned to the laboratory after transplantation and repeatedly washed. An average of 22 x106 nucleated cells could be recovered, yielding a mean 473 x106 CD3/CD56+ cells at the end of the culture period (1485 fold expansion of CIK cells). CIK cells generated from CB showed strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias (42–72% killing at a 30:1 E:T ratio). More importantly, they were cytotoxic against AML blasts isolated from 2 patients (41% lysis). During expansion CB derived CIK cells upregulated the NKG2D marker from a mean fluorescence intensity of 49 to 209. Furthermore they expressed perforin and granzyme molecules in >90% of cells. These observations open up the possibility of a future clinical application of this protocol, performed in GMP conditions. Patients relapsing following cord blood transplantation may be treated with CIK cells expanded from the same cord blood unit, where donors would not be anymore available for cell mediated immunotherapy.