Tumor RNA-pulsed Dendritic Cells Research Articles

Immunotherapy reverses glioma-driven dysfunction of immune system homeostasis

BackgroundGlioma-induced immune dysregulation of the hematopoietic system has been described in a limited number of studies. In this study, our group further demonstrates that gliomas interrupt the cellular differentiation programming...

Total tumor RNA pulsed dendritic cells plus adoptive transfer of ex-vivo enriched autologous T-lymphocytes in the treatment of children with primary brain tumors

Total tumor RNA pulsed dendritic cells plus adoptive transfer of ex-vivo enriched autologous T-lymphocytes in the treatment of children with primary brain tumors

IMPS-07T CELL RECEPTOR VARIABLE β CHAIN (TCR Vβ) USAGE TO PREDICT AND GENERATE TUMOR SPECIFIC T LYMPHOCYTES AGAINST INVASIVE GLIOMAS

INTRODUCTION: Adoptive cellular therapy has been demonstrated to be a highly efficacious immunotherapy against solid tumors. In this study, we predict T cell populations with high affinity to glioma and generate a T cell population highly enriched for tumor-reactive T cells. OBJECTIVES: Major barriers to therapeutic development against brain tumors are that brain tumors are highly heterogeneous and tumor antigens are largely uncharacterized. The primary objective of this study was to predict and generate a polyclonal population of T lymphocytes that is highly enriched for tumor-specific T cells and overcomes these limitations. METHODS: We spectratyped the TCR Vβ repertoire of glioma infiltrating CD3+ cells in order to identify T cell populations with high affinity to tumor. Based on the identified TCR Vβ family expression, we isolated and reliably expanded these T cells from splenocytes of mice previously immunized with tumor RNA-pulsed dendritic cells. These cells were adoptively transferred into intracranial glioma bearing mice followed by dendritic cell vaccine. RESULTS: In vitro functionality assays demonstrated that T cells with TCR Vβ families predicted to have high affinity to tumors were responsible for anti-tumor immunity. Adoptive cellular therapy using T cells expressing TCR Vβ 5.1, 5.2, TCR Vβ 6, and TCR Vβ 8.1, 8.2 against intracranial tumor led to cures in 60% of treated mice. We further identify that the in vivo expansion of T cells expressing the TCR Vβ 6 family is specifically associated with long term survival. Failure of the persistence of these T cells is directly associated with tumor escape. CONCLUSION: Our methods allow us to generate highly tumor-reactive polyclonal T cell populations without the need for identifying tumor antigens. We are currently employing these analyses in clinical trials of adoptive cellular therapy targeting pediatric and adult gliomas under development at our center (FDA IND BB-14058).

IM-15 * ANTI-TUMOR EFFICACY OF ADOPTIVE CELLULAR THERAPY IS SIGNIFICANTLY INCREASED BY HEMATOPOIETIC STEM CELLS

Immunotherapeutic approaches against solid tumors can be curative with a demonstrably high degree of specificity, and adoptive T cell therapies have proven to be efficacious platform against metastatic disease. Superior clinical outcomes in response to adoptive cellular therapies are correlated with anti-tumor function, intratumoral migration and persistence of adoptively transferred tumor-specific T cells at the tumor site. Adoptive cellular therapy after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has been largely limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other solid cancers. We have employed tumor RNA-pulsed dendritic cells to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative adoptive cellular therapy in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. We have identified the TCR Vb clone largely responsible for immunologic rejection of intracranial murine glioma. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative conditioning, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed dendritic cell (DC) vaccines. Hematopoietic stem cells (HSCs), administered for salvage from myeloablative conditioning, rapidly migrated to areas of intracranial tumor growth and facilitated the recruitment of tumor-specific lymphocytes through HSC-elaborated chemokines and enhanced immunologic rejection of intracranial tumors during adoptive cellular therapy. In addition, we found that HSCs mediate a significant expansion of tumor-specific T cells under both myeloablative and non-myeloablative host conditioning. The expansion of tumor-specific T cells expressing specific TCR Vβ clones in intracranial tumor bearing mice is associated with tumor rejection. Our data indicate novel roles for the use of HSCs in the immunologic treatment of malignant gliomas and possibly other solid tumors.

Novel role of hematopoietic stem cells in immunologic rejection of malignant gliomas

Adoptive cellular therapy (ACT) after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has been largely limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other solid cancers. We have employed tumor RNA-pulsed dendritic cells (DCs) to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative ACT in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative (MA) conditioning, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed DC vaccines. Hematopoietic stem cells (HSCs), administered for salvage from MA conditioning, rapidly migrated to areas of intracranial tumor growth and facilitated the recruitment of tumor-specific lymphocytes through HSC-elaborated chemokines and enhanced immunologic rejection of intracranial tumors during ACT. Furthermore, HSC transplant under non-myeloablative (NMA) conditions also enhanced immunologic tumor rejection, indicating a novel role for the use of HSCs in the immunologic treatment of malignant gliomas and possibly other solid tumors.

IT-10 * SYNERGISTIC CELLULAR INTERACTIONS IN ADOPTIVE IMMUNOTHERAPY LEADS TO IMMUNOLOGIC REJECTION OF MALIGNANT GLIOMA

Adoptive cellular therapy after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has previously been limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other cancers. We demonstrate that tumor RNA-pulsed dendritic cells can be used to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative adoptive cellular therapy in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative conditioning and hematopoietic stem cell transfer, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed dendritic cell (DC) vaccines. By employing this platform we activate a polyclonal population of tumor reactive T cells and have further identified tumor-specific repertoires of tumor-reactive CD8+ T lymphocytes. Furthermore, we have observed a significant increase of clonotypic frequency of tumor-specific CD8+ cells by following TCR β chain expression in mice that are cured of established tumor by adoptive cellular therapy, indicating the ability to potentially predict treatment outcomes in a preclinical setting. Clinical studies exploring the feasibility and potential survival benefit of this platform against both pediatric and adult CNS malignancies are currently underway in our institution. Our adoptive cellular therapy platform has demonstrated superior immunologic treatment of malignant gliomas and holds potential against other solid tumors.

Abstract 4752: Induction of cytotoxic T lymphocytes primed with tumor RNA-loaded dendritic cells in esophageal squamous cell carcinoma: Preliminary step for DC vaccine design

Abstract Backgrounds: Dendritic Cells (DC) are potent antigen presenting cells with the ability to prime naïve T cells and convert them to cytotoxic CTL. We evaluated the capability of autologous DCs transfected with total Tumor and normal RNA to induce cytotoxic CTL as the preliminary step to design DC-based vaccine in the esophageal squamous cell carcinoma (ESCC). Methods: Monocytes-derived DCs were electroporated with either total tumor RNA or normal RNA. T cells were then primed with tumor RNA transfected DCs and lysing effects of the generated CTL were measured with Cytotoxicity assay and IFN-γ Release Elispot Assay. Results: Cytotoxicity against DCs loaded with tumoral RNA was induced (%24.8±5.2 SEM) while in normal RNA-loaded DCs it was minimal (%6.1±2.4 SEM) and significantly lower (p < 0.05). INF-γ secretion was more than 2-folds higher in tumoral RNA-loaded DCs when compared with normal RNA-loaded DCs (p < 0.05). Conclusion: Electroporating DCs with tumor RNA made tumor antigen presenting cells that enhanced cytotoxicity effects of T cells against ESCC. This may be a useful autologous ex vivo screening tool for confirming the lytic effects of primed T cells on tumor and evaluate probable further adverse effects on noncancerous tissue. These data are a crucial preliminary step to establish a total tumor RNA-pulsed DC vaccine therapy of ESCC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4752.

Enhanced Inhibition of Tumor Growth With Depletion of CD25 Regulatory Cells and Intratumoral Immunization With Tumor RNA-Pulsed Dendritic Cells in a C57BL/6 Pancreatic Tumor Model

Enhanced Inhibition of Tumor Growth With Depletion of CD25 Regulatory Cells and Intratumoral Immunization With Tumor RNA-Pulsed Dendritic Cells in a C57BL/6 Pancreatic Tumor Model

Enhanced Antitumor Effects by the Coculture of Allotumor RNA-Pulsed Dendritic Cells with Autologous Cytokine-Induced Killer Cells on Hormone-Refractory Prostate Cancer

In this study, we evaluated antitumor effects of allotumour RNA-transfected dendritic cells (DCs) cocultured with autologous cytokine-induced killer cells (CIKs) on hormone-refractory prostate cancer. The cocultured cells enhanced prostate cancer cytolysis from 26% (CIKs-induced cytolysis) to 80.8%. They also increased the productions of CD4+ Th1 (IFN-γ+IL-4−, 55.52%) and CD8+ T (IFN-γ+, 69.59%) cells determined by intracellular cytokines IFN-γ /IL-4 staining and reduced the rate of CD4+ CD25+ cells from 18.72% (in CIKs) to 9.72%. The cocultured cells significantly inhibited tumor growth in SCID mouse and induced cancer cells necrosis and apoptosis. Our study indicates that tumor RNA-pulsed DCs cocultured with autologous CIKs significantly enhance antitumor immunity, which can be induced by increased CD4+ Th1 and CD8+ T cells and decreased CD4+CD25+ regulatory T (Treg) cells. This provides a potential immunotherapy strategy for HRPC.

Effective osteosarcoma cytolysis using cytokine-induced killer cells pre-inoculated with tumor RNA-pulsed dendritic cells

Osteosarcoma with distant metastases at late stage has posed a challenge for novel therapeutic modalities. The application of cytokine-induced killer (CIK) cells to osteosarcoma constitutes a promising strategy. This approach had been studied in multiple myeloma and breast cancers, where CIK cells exhibited specific cytotoxicity toward malignant cells while sparing wild-type tissues. However, the consistency of CIK cell-induced anti-tumor cytotoxicity has not been thoroughly examined. We investigated whether autologous CIK cells could effectively induce cytolysis of cultured osteosarcoma cells. In addition to the observed CIK cell-induced osteosarcoma cytolysis, the pre-incubation of CIK cells with autologous dendritic cells pulsed with tumor's total RNA further enhanced the tumor cytolysis to greater than 6-fold. The anti-tumor cytolysis was optimized in complete autologous setting, and was attenuated with allogeneic components. The advantage of the co-culture with RNA-pulsed DC was lost when high CIK cell density was employed for anti-tumor cytotoxic assay, but was maintained in purified CD3(+)CD56(+) cells isolated from the CIK cells. This finding implied that CIK cells at limited cell density could induce effective osteosarcoma cytolysis with an aid from tumor antigen presentation on dendritic cell surface.

Effective osteosarcoma cytolysis using cytokine-induced killer cells pre-inoculated with tumor RNA-pulsed dendritic cells

Osteosarcoma with distant metastases at late stage has posed a challenge for novel therapeutic modalities. The application of cytokine-induced killer (CIK) cells to osteosarcoma constitutes a promising strategy. This approach had been studied in multiple myeloma and breast cancers, where CIK cells exhibited specific cytotoxicity toward malignant cells while sparing wild-type tissues. However, the consistency of CIK cell-induced anti-tumor cytotoxicity has not been thoroughly examined. We investigated whether autologous CIK cells could effectively induce cytolysis of cultured osteosarcoma cells. In addition to the observed CIK cell-induced osteosarcoma cytolysis, the pre-incubation of CIK cells with autologous dendritic cells pulsed with tumor’s total RNA further enhanced the tumor cytolysis to greater than 6-fold. The anti-tumor cytolysis was optimized in complete autologous setting, and was attenuated with allogeneic components. The advantage of the co-culture with RNA-pulsed DC was lost when high CIK cell density was employed for anti-tumor cytotoxic assay, but was maintained in purified CD3 +CD56 + cells isolated from the CIK cells. This finding implied that CIK cells at limited cell density could induce effective osteosarcoma cytolysis with an aid from tumor antigen presentation on dendritic cell surface.

Dendritic cells pulsed with pancreatic cancer total tumor RNA generate specific antipancreatic cancer T cells.

RNA-based dendritic cell immunotherapy with the use of total tumor RNA provides the potential to generate a polyclonal immune response to multiple known and unknown tumor antigens without HLA restriction. Our study evaluated this approach as potential immunotherapy for patients with pancreatic cancer. Dendritic cells were generated using adherent monocytes isolated from peripheral blood of patients with pancreatic cancer and evaluated phenotypically by flow cytometry to determine whether dendritic cells could be generated from the blood of patients with pancreatic cancer. Immature dendritic cells were transfected with mRNA encoding full-length carcinoembryonic antigen (CEA) or pancreatic cancer total tumor messenger RNA, and then matured. Matured dendritic cell phenotypes were also analyzed by flow cytometry. Transfected, matured dendritic cells were used to stimulate autologous T cells, and the resultant antigen-specific effector T cells were analyzed by interferon-gamma Elispot assay. Immature dendritic cells with characteristic phenotypic markers CD40, CD80, and CD86 were successfully isolated from the blood of patients with pancreatic cancer. Incubation with maturation agents increased expression of CD80 and CD83, demonstrating the induction of a mature antigen-presenting phenotype. Dendritic cells transfected with a pancreatic cancer-associated antigen (CEA) generated antigen-specific T cells (P<0.05). Dendritic cells transfected with autologous total tumor pancreatic cancer RNA generated T cells that specifically recognized HLA-matched pancreatic cancer cell lines (P<0.05 compared to control cell lines). Dendritic cells from patients with pancreatic cancer maintain the ability to translate and process transfected RNA and serve as mature antigen-presenting cells. These RNA-transfected dendritic cells from pancreatic cancer patients successfully generate specific T cells against the pancreatic cancer-associated antigen CEA as well as T cells that specifically recognize pancreatic cancer cells. These data suggest that total tumor RNA-pulsed dendritic cells may have potential as an adjuvant immunotherapy for patients with pancreatic cancer.

Antitumor effects of vaccine consisting of dendritic cells pulsed with tumor RNA from gastric cancer.

To investigate the immunotherapeutic potential of vaccine consisting of dendritic cells (DCs) pulsed with total RNA from MFC gastric cancer cells. DCs were prepared from the spleens of strain 615 mice by magnetic cell sorting (MACS). After culture for 24 h, DCs were pulsed with total RNA from MFC gastric cancer cells. Mice of one group were immunized with tumor RNA pulsed DC (RNA/DC) at the dosage of 1X10(6) on d 14 and 7 by s c inoculation before tumor implantation. Mice of another group were immunized with unpulsed DC (UDC) at the same dosage on days as the RNA/DC group. The third group of control mice was untreated. On d 0, all the mice were challenged with s c injections of 5X10(5) MFC gastric cancer cells. After inoculation, the mice were monitored closely with respect to tumor growth. Activities of NK cells in PBL and splenocytes and CTL were tested. On d 21 after tumor cell inoculation, the mice of control group manifested the largest tumors with volume at a mean of 2.6323+/-1.1435 cm(3), followed by the UDC and RNA/DC groups with mean volumes at 0.7536+/-0.3659 cm(3) and 0.3688+/-0.6571 cm(3), respectively. The activities of NK cells in PBL and splenocytes in RNA/DC group were 66.2% and 65.4%, respectively, higher than that in the control group. The tumor specific CTL activity in RNA/DC group was 49.5%, higher than that in the control group. The tumor vaccine with DCs pulsed with total RNA from gastric cancer cells possesses the ability to stimulate tumor specific CTL activity and to establish anti-tumor immunity when administered in vivo.

Enhanced therapeutic efficacy of tumor RNA-pulsed dendritic cells after genetic modification with lymphotactin.

Pulsing dendritic cells (DCs) with tumor cell-derived mRNA is regarded as an attractive alternative in the development of DC-based tumor vaccines. Our aim is to improve the therapeutic efficacy of DC-based tumor RNA vaccines by augmenting the preferential chemotaxis of DCs to T cells. Mouse bone marrow-derived DCs were genetically modified with lymphotactin (Lptn) by adenovirus vector, which conferred on DCs preferential chemotaxis to CD4+ and CD8+ T cells (Cao et al., 1998). Lptn gene-modified DCs (Lptn-DCs) were pulsed with tumor mRNA and used for vaccination in the tumor models of 3LL lung carcinoma and B16 melanoma. In both tumor models, immunization with 4 X 10(4) tumor RNA-pulsed Lptn-DCs induced more potent CTL activity, compared with their counterparts, specifically against tumor cells and Mut1 or tyrosinase-related protein 2 (TRP-2) peptide-pulsed RMA-S cells, and rendered the immunized mice resistant to tumor challenge much more effectively. CD8+ T cells were necessary and sufficient to generate the protection of Lptn-DC-based RNA tumor vaccines, and CD4+ T cells were required for the induction of tumor rejection. In the preestablished 3LL and B16 tumor models, vaccination with DC-based or LacZ-DC-based tumor RNA vaccines (2 X 10(5) cells) could reduce pulmonary metastasis and extend survival of tumor-bearing mice, but was less effective than the Lptn-DC counterpart (with 60-80% mice surviving). When the immunizing dose was decreased to 4 X 10(4) cells, Lptn-DC-based tumor vaccines rather than their counterparts were still significantly effective. Our studies provide a potential strategy to improve the efficacy of DC-based vaccines, and a new approach to immunological intervention by chemokines.