Induction Of Cytotoxic T Lymphocytes Research Articles

EWS-FLI-1-Targeted Cytotoxic T-cell Killing of Multiple Tumor Types Belonging to the Ewing Sarcoma Family of Tumors

The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types. Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.

Immunosuppressive effect of regulatory T lymphocytes in lung cancer, with special reference to their effects on the induction of autologous tumor-specific cytotoxic T lymphocytes

It is not easy to induce cytotoxic T lymphocytes (CTLs) against cancer in in vitro culture. Regulatory T cells (Tregs) are considered to play a pivotal role in tumor immune escape. In this study, we analyzed the distribution of Tregs among tumor-infiltrating lymphocytes (TILs), regional lymph node lymphocytes (RLNLs) and peripheral blood lymphocytes (PBLs) in patients with lung cancer, and analyzed the effect of Tregs on the induction of CTLs in vitro. A total of 84 patients with non-small cell lung cancer underwent surgery between January 2003 and December 2004. The TILs, RLNLs and PBLs from these patients were subjected to a comparison analysis. The proportion of CD4(+)CD25(+)Foxp3(+) cells in these lymphocytes was determined by flow cytometry. The effects of Tregs on the induction of CTLs was analyzed by the depletion of Tregs in mixed lymphocyte-tumor cell culture (MLTC). The average proportions of Tregs in the TILs, RLNLs and PBLs were 10.4±9.5, 4.4±2.4 and 2.8±2.1%, respectively. The proportion of Tregs in the RLNLs was significantly higher than that in the PBLs (P<0.001); furthermore, TILs contained a larger number of Tregs than RLNLs (P=0.034). These Tregs substantially suppressed the induction of CTLs against autologous tumor cells. The depletion of Tregs in the MLTC resulted in the successful induction of CTLs. Tregs were found at a higher frequency in the TILs and RLNLs than in the PBLs in lung cancer patients. Since Tregs inhibited the induction of CTLs, the depletion of Tregs may represent a new therapeutic strategy for lung cancer patients.

Repetitive peptide boosting progressively enhances functional memory CTLs

Cytotoxic T lymphocytes (CTLs) play a critical role in controlling intracellular pathogens and cancer cells, and induction of memory CTLs holds promise for developing effective vaccines against critical virus infections. However, generating memory CTLs remains a major challenge for conventional vector-based, prime-boost vaccinations. Thus, it is imperative that we explore nonconventional alternatives, such as boosting without vectors. We show here that repetitive intravenous boosting with peptide and adjuvant generates memory CD8 T cells of sufficient quality and quantity to protect against infection in mice. The resulting memory CTLs possess a unique and long-lasting effector memory phenotype, characterized by decreased interferon-γ but increased granzyme B production. These results are observed in both transgenic and endogenous models. Overall, our findings have important implications for future vaccine development, as they suggest that intravenous peptide boosting with adjuvant following priming can induce long-term functional memory CTLs.

Identification of novel MAGE-A6- and MAGE-A12-derived HLA-A24-restricted cytotoxic T lymphocyte epitopes using an in silico peptide-docking assay.

Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called α-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24(+) melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.

A membrane vesicle-based dual vaccine against melanoma and Lewis lung carcinoma

In the past few years, cell-derived membrane vesicle-based tumor vaccines have been considered as valuable new tools for cancer immunotherapy. Despite promising results in cancer clinical trails, an improved method is urgently needed for high efficiency tumor vaccines for a broad spectrum of tumors. Here we developed a single membrane vesicle-based vaccine, which is active in repressing both melanoma (B16) and Lewis lung carcinoma (LLC) tumor growth. By using the intrinsic function of dendritic cells in the processing and presentation of antigens, we generated dendritic cell (DC)-derived membrane vesicles (DC-mv) bearing tumor antigens from both B16 and LLC cells. Vaccination with this DC-mv-based dual vaccine induced specific cytotoxic T lymphocytes (CTL)-dependent tumor rejection and suppressed the growth of both types of tumor xenografts in mice. In addition, induction of CTL by this vaccine resulted in cross-protection responses and consequently enabled significant enhanced anti-tumor effects, indicating the synergistic anti-tumor activity. Our study suggests that the DC-mv-based vaccine holds great potential as a highly effective, versatile, cell-free vaccine for inhibition of multiple types of tumor growth.

Lymphocyte-polarized dendritic cells are highly effective in inducing tumor-specific CTLs

High activity of dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) led to their application as therapeutic cancer vaccines. The ability of DCs to produce IL-12p70 is one of the key requirements for effective CTL induction and a predictive marker of their therapeutic efficacy in vivo. We have previously reported that defined cocktails of cytokines, involving TNFα and IFNγ, induce mature type-1 polarized DCs (DC1s) which produce strongly elevated levels of IL-12 and CXCL10/IP10 upon CD40 ligation compared to “standard” PGE2-matured DCs (sDCs; matured with IL-1β, IL-6, TNFα, and PGE2) and show higher CTL-inducing activity. Guided by our observations that DC1s can be induced by TNFα- and IFNγ-producing CD8+ T cells, we have tested the feasibility of using lymphocytes to generate DC1s in a clinically-compatible process, to limit the need for clinical-grade recombinant cytokines and the associated costs. CD3/CD28 activation of bulk lymphocytes expanded them and primed them for effective production of IFNγ and TNFα following restimulation. Restimulated lymphocytes, or their culture supernatants, enhanced the maturation status of immature (i)DCs, elevating their expression of CD80, CD83 and CCR7, and the ability to produce IL-12p70 and CXCL10 upon subsequent CD40 ligation. The “lymphocyte-matured” DC1s showed elevated migration in response to the lymph-node-directing chemokine, CCL21, when compared to iDCs. When loaded with antigenic peptides, supernatant-matured DCs induced much high levels of CTLs recognizing tumor-associated antigenic epitope, than PGE2-matured DCs from the same donors. These results demonstrate the feasibility of generation of polarized DC1s using autologous lymphocytes.

Abstract LB-431: Augmented induction of tumor antigen-specific CTL by cetuximab in head and neck cancer patients does not correlate with Fcγ receptor IIIa genotype, but relies on NK:DC cross talk mediated by interferon-γ and NKG2D

Abstract Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ∼20% of HNC patients are clinically responsive to treatment with the EGFR-specific monoclonal antibody (mAb) cetuximab. To date, no reliable biomarker of clinical efficacy has been identified to predict clinical response to cetuximab therapy in HNC. Modest, yet statistically significant correlations of Fcγ receptor (FcγR) IIIa polymorphisms with clinical outcome have been noted in B cell lymphoma, breast, and colorectal cancer patients treated with rituximab, trastuzumab, and cetuximab, respectively. Therefore, we have investigated whether a known FcγRIIIa polymorphism in natural killer (NK) cells at amino acid position 158 (valine [V] vs. phenylalanine [F]) correlates with the anti-tumor activity of cetuximab in immunodeficient mice engrafted with human HNC tumors as well as with the induction of EGFR-specific cytotoxic T lymphocytes (CTL) and clinical course in HNC patients treated with cetuximab. When injected into immunodeficient mice, human NK cells expressing the V allele at this position demonstrated significantly greater ability to control the growth of xenografted HNC tumors. However, we did not find a significant correlation between FcγRIIIa genotype and disease free survival (p=0.683) in a cohort of 107 consecutive HNC patients treated with regimens incorporating cetuximab. Furthermore, we demonstrate for the first time that in the presence of cetuximab-opsonized HNC cells, NK cells trigger maturation of dendritic cells (DC), leading to augmented TA-specific cross-priming of EGFR and MAGE-specific CTL in vitro, and in vivo in cetuximab-treated HNC patients. The significantly higher frequencies of EGFR-specific CTL we found in HLA-A2+ PBMC from cetuximab-treated HNC patients relative to cetuximab-naive HNC patients (p&amp;lt;0.001) also did not correlate with FcγRIIIa genotype, but their induction was dependent on NK:DC cross talk mediated by interferon-γ and NKG2D. Taken together, our data indicate that cetuximab treatment augments the priming of both EGFR and non-EGFR TA-specific cellular immunity in vivo, thereby providing a novel immune mechanism for enhanced anti-tumor activity of TA-specific mAb relevant to clinical response, as well as a potential biomarker for monitoring this response in mAb-treated cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-431. doi:1538-7445.AM2012-LB-431

Abstract 510: Identification and characterization of immunogenic epitopes from CD133 and their potential for use to immunologically target cancer stem cells

Abstract CD133 is a marker that identifies cancer stem cells (CSC) on many solid tumors and its expression has been correlated with shortened survival. Although expressed on many normal tissues and stem cells, CD133 is overexpressed on CSC and normal SC are negative or express low levels of MHC making them less sensitive to recognition by CD8+ cytotoxic T lymphocytes (CTL). Potential CTL epitopes were identified by algorithms to predict binding to HLA-A2. Binding studies showed high affinity binding and low off rates for four candidate epitopes. CTL induction studies using IL-12 secreting dendritic cells to stimulate CD8+ T cells evaluated the immunogenicity of the candidate peptides after three or four weekly in vitro stimulations. Multimer and IFNγ responses were observed with CD133-753, 405 and 708 epitopes. Subsequent studies demonstrated killing of T2 cells loaded with peptide. To evaluate the potential for autoimmunity, mouse homolog peptides of the 753 and 405 epitopes that were shown to have high affinity binding to human HLA-A2 were used to immunize HLA-A2 transgenic mice (9CB6F1-Tg (HLA-A*0201/H2-Kb) A*0201). Mice were immunized 3 times at 3 week intervals in IFA + HBV helper peptide and spleens were harvested 2 weeks after the last immunization and stimulated in vitro for one week with peptide pulsed LPS stimulated- APC. An additional group was sacrificed 6 weeks after the last immunization and studied. IFNγ ELISpot assays showed immune responses to the 753 and 405 peptides in 35% and 40% of mice. Organs, including heart, lung, liver, kidney, stomach, intestine, brain, bone marrow, gonads, and eyes from mice with immune responses were found to be negative for lymphocytic infiltrations supporting a lack of autoimmunity related to the immune response to these peptides. Together these studies support the safety and immunogenicity of these peptides as a potential vaccine to target CD133 cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 510. doi:1538-7445.AM2012-510

Enhanced immunity against hepatoma induced by dendritic cells pulsed with Hsp70-H22 peptide complexes and CD40L

Dendritic cell (DC)-based cancer vaccines have become an attractive antitumour therapeutic approach. However, clinical application of current DC-based cancer vaccines has been limited by their ineffectiveness. Heat shock protein 70 from Mycobacterium tuberculosis (TBhsp70) is known to have a potent adjuvant capability to induce maturation of DCs and thus acts as an alternative ligand to the CD40 ligand (CD40L) on T cells to induce a T-cell response. The aim of this study is to investigate whether the combination of TBhsp70-H22 tumour-peptide complexes and CD40L might improve the antitumour efficacy for development of therapeutic DC-based vaccines against hepatoma. The CD40, CD80, CD86 and HLA-DR expression on DCs pulsed with TBhsp70-H22 tumour-peptide complexes and soluble CD40L was studied by flow cytometric analysis, and T-helper type 1 cytokine secretion, such as IL-12p70 secretion, was tested by ELISA. The H22-specific cytotoxic T-lymphocytes (CTLs) were detected by a (51)Cr-release assay, and the in vivo antitumour immunity against hepatoma was measured by utilising H22-tumour-bearing mice after therapeutic administration. Up-regulation of CD40, CD80, CD86 and HLA-DR expression on DCs pulsed with TBhsp70-H22 tumour-peptide complexes and CD40L was found, which stimulated a high level of T-helper type 1 cytokine secretion, such as IL-12p70, and resulted in the induction of H22-specific CTLs. The therapeutic administration of DCs pulsed in vitro with TBhsp70-H22 tumour-peptide complexes and CD40L significantly reduced the progression of H22 tumours in mice compared with DC-Hsp70-H22 peptide complexes or DC-CD40L alone. Our findings demonstrate that DCs pulsed with Hsp70-H22-peptide complexes and CD40L enhance the antitumour immunity against hepatoma, which provides a novel immunotherapeutic approach against cancer.

Cytotoxic T lymphocytes from cattle immunized against Theileria parva exhibit pronounced cross-reactivity among different strain-specific epitopes of the Tp1 antigen

The protozoan parasite Theileria parva causes a usually fatal disease in cattle, known as East Coast fever. Cattle can be vaccinated by injecting live parasites simultaneously with long acting oxytetracycline (the infection and treatment method, ITM). The immunity induced by ITM is believed to be mediated by cytotoxic T lymphocytes (CTL). Although effective, the ITM vaccine has disadvantages such as the need for a liquid nitrogen cold chain and a complex production process, which may be overcome by the development of a subunit vaccine. However, the high level of antigenic polymorphism among different strains of T. parva may hinder the development of a subunit vaccine aimed at induction of a protective CTL response.In this study, the CTL cross-reactivity among T. parva strains was examined. The Tp1214–224 epitope has previously been shown to be recognized by cattle of the A18 BoLA type. Three different variants of this epitope have been identified from different T. parva strains. Here, bulk CTL and CTL clones were generated from two animals using both the live sporozoite vaccine composed of three different strains and a Muguga strain for immunization. The cross-reactivity of these CTL with the three variant Tp1 epitopes was examined in interferon gamma ELISPOT assays and CTL killing assays.CD8+ cells from both animals cross-reacted with the three variant CTL epitopes in interferon gamma ELISPOT assays, although the CD8+ cells from the Muguga-immunized animal showed a more epitope restricted response. Clones from the vaccine immunized animal showed diverse response patterns with clones responding to each variant peptide. Although some variability in the cytotoxic response was observed, overall strong cross-reactivity among the variant Tp1 epitopes was seen in both animals. Such epitope polymorphism does not, in this case, serve as a potential challenge in a putative subunit vaccine as it would be sufficient to only include one of the variant epitopes.

Human dendritic cells sequentially matured with CD4+ T cells as a secondary signal favor CTL and long-term T memory cell responses

Dendritic cells (DCs) are professional antigen-presenting cells involved in the control and initiation of immune responses. In vivo, DCs exposed at the periphery to maturation stimuli migrate to lymph nodes, where they receive secondary signals from CD4+ T helper cells. These DCs become able to initiate CD8+ cytotoxic T lymphocyte (CTL) responses. However, in vitro investigations concerning human monocyte-derived DCs have never focused on their functional properties after such sequential maturation. Here, we studied human DC phenotypes and functions according to this sequential exposure to maturation stimuli. As first signals, we used TNF-α/polyI:C mimicking inflammatory and pathogen stimuli and, as second signals, we compared activated CD4+ T helper cells to a combination of CD40-L/ IFN-γ. Our results show that a sequential activation with activated CD4+ T cells dramatically increased the maturation of DCs in terms of their phenotype and cytokine secretion compared to DCs activated with maturation stimuli delivered simultaneously. Furthermore, this sequential maturation led to the induction of CTL with a long-term effector and central memory phenotypes. Thus, sequential delivery of maturation stimuli, which includes CD4+ T cells, should be considered in the future to improve the induction of long-term CTL memory in DC-based immunotherapy.

Liposomes and nanotechnology in drug development: focus on oncotargets

Nanotechnology is the development of an engineered device at the atomic, molecular, and macromolecular level in the nanometer range. Advances in nanotechnology have proven beneficial in therapeutic fields such as drug-delivery and gene/protein delivery. Antigen delivery systems are important for inducing and modifying immune responses. In cellular immunity, cytotoxic T lymphocytes (CTLs) are important in the host defense against tumors. Key to the development of CTL-inducible vaccines is the ability to deliver antigens to antigen-presenting cells efficiently and to induce the subsequent activation of T cell-mediated immunity without adjuvants, as they can induce excessive inflammation leading to systemic febrile disease. Since expression and cloning methods for tumor-associated antigens have been reported, cancer vaccines that induce effective cell immunity may be promising therapeutic candidates, but Th2 cells are undesirable for use in cancer immunotherapy. Peptide vaccines have immunological and economic advantages as cancer vaccines because CTL epitope peptides from tumor-associated antigens have high antigen-specificity. However, cancer vaccines have had limited effectiveness in clinical responses due to the ability of cancer cells to “escape” from cancer immunity and a low efficiency of antigen-specific CTL induction due to immunogenic-free synthetic peptides. In contrast, carbohydrate-decorated particles such as carbohydrate-coated liposomes with encapsulated antigens might be more suitable as antigen delivery vehicles to antigen-presenting cells. Oligomannose-coated liposomes (OML) can eliminate established tumors in mouse cancer models. In addition, OMLs with an encased antigen can induce antigen-specific CTLs from peripheral blood mononuclear cells obtained from patients. Feasibility studies of OML-based vaccines have revealed their potential for clinical use as vaccines for diseases where CTLs act as effector cells. Furthermore, use of the hepatitis B core particle, in which tumor-antigen epitopes are set, has consistently been shown to induce strong CTL responses without the use of an adjuvant. Thus, nanoparticles may provide a new prophylactic strategy for infectious disease and therapeutic approaches for cancer via the induction of T-cell immunity.

Immunotherapy Synergizes with Chemotherapy Targeting Pancreatic Cancer

Pancreatic adenocarcinoma is a devastating disease and has an extremely poor prognosis. Despite aggressive treatment approaches, including surgery, chemotherapy and radiation, the overall 5‐year survival rate is less than 5%. Therefore, the development of therapeutic strategies for advanced pancreatic cancer has traditionally been considered particularly challenging to improve clinical outcomes. Although immunotherapy that is designed to target tumor-associated antigens (TAAs) is a promising treatment approach, immunotherapy alone is limited by the number of cytotoxic T lymphocytes (CTLs) able to penetrate the large established pancreatic tumor. Even if large numbers of antigen-specific polyclonal CTLs were generated in vitro and injected into the patients, CTLs cannot penetrate into tumor sites because of stroma cells such as cancer-associated fibroblasts, tolerogenic dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), immunosuppressive tumor-associated macrophages and Tregs. Moreover, these cells produce immunosuppressive cytokines such as IL-10 and TGF-β; thus, clinical responses by immunotherapy alone cannot induce efficient antitumor immunity in patients with advanced pancreatic cancer. On the other hand, cytotoxic chemotherapy is well known to blunt immune responses, because of its toxicity for dividing cells including peripheral lymphoid tissue as well as the bone marrow. However, increasing evidence has been mounting to suggest that immunotherapy has the possibility of achieving better success when used in combination with chemotherapy [1]. For example, necrotic or apoptotic tumor cells induced by chemotherapy can be phagocytosed by DCs that are potent antigen-presenting cells, processed and presented to immune lymphocytes, followed by induction of antitumor immune responses. Different chemotherapeutic agents may kill tumor cells through an apparently homogeneous apoptotic pathway. Of note, treatment of pancreatic cancer cells with a standard cytotoxic agent for pancreatic cancer, gemcitabine, results in enhanced cross-presentation of TAAs by DCs and CTL induction [2]. Moreover, gemcitabine can also inhibit Tregs, B cells and MDSCs [2,3], but induce the proliferation of DCs [4]. These phenomena suggest that gemcitabine induces efficient CTL responses, improves the penetration of CTLs into the tumor parenchyma, and enhances tumor cell sensitivity to lyze antigen-specific CTLs. Thus, immunotherapy may mediate a potent antitumor effect when c ombined with chemotherapy.

High‐capacity adenoviral vectors circumvent the limitations of ΔE1 and ΔE1/ΔE3 adenovirus vectors to induce multispecific transgene product‐directed CD8 T‐cell responses

The ability to induce cytotoxic T lymphocyte (CTL) responses that are multispecific is considered to comprise an essential feature for an efficacious genetic vaccine against many pathogens including HIV and hepatitis C virus. ΔE1Ad vectors are promising vectored vaccines but have been shown to induce antigen-specific CTLs with only limited multispecificity. In the present study, we investigated the applicability of gene-deleted high-capacity adenovirus (HC-Ad) vectors and focused on the induction of multispecific CTL responses. We generated Δ E1 and HC-Ad vectors expressing hepatitis B virus small surface antigen (HBsAg). We comparatively analyzed the CTL profiles against various transgene product- and vector-derived epitopes in several mouse strains and HBsAg- and vector-directed antibody responses. HC-Ad vectors efficiently induced multispecific HBsAg-directed CTLs. By contrast, ΔE1Ad vectors mainly primed CTLs against one immunodominant epitope of HBsAg. This absence of multispecific CTL responses correlated with the induction of CTLs against viral epitopes generated by de novo expression of Ad genes from the ΔE1Ad vector. However, Ad-specific CTLs induced in trans did not impair HC-AdS-induced multispecific CTL responses against HBsAg. Finally, HC-Ad vectors also induced higher HBsAg antibody titers compared to ΔE1Ad vectors. De novo expression of viral genes from ΔE1Ad vector genomes restricts the multispecificity of transgene product-specific CTLs by immunodominance effects. HC-Ad vectors devoid of Ad genes are favorable for the induction of both multispecific CD8 T-cell responses and high antibody responses. Our results suggest the deletion of Ad genes as an important means for developing potent Ad-based vectored vaccines.

Enhanced tumor immunity of WT1 peptide vaccination by interferon-β administration

To induce and activate tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) for cancer immunity, it is important not only to select potent CTL epitopes but also to combine them with appropriate immunopotentiating agents. Here we investigated whether tumor immunity induced by WT1 peptide vaccination could be enhanced by IFN-β. For the experimental group, C57BL/6 mice were twice pre-treated with WT1 peptide vaccine, implanted with WT1-expressing C1498 cells, and treated four times with WT1 peptide vaccine at one-week intervals. During the vaccination period, IFN-β was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-β alone, or PBS alone. The mice in the experimental group rejected tumor cells and survived significantly longer than mice in the control groups. The overall survival on day 75 was 40% for the mice treated with WT1 peptide+IFN-β, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-β alone or PBS alone, respectively. Induction of WT1-specific CTLs and enhancement of NK activity were detected in splenocytes from mice in the experimental group. Furthermore, administration of IFN-β enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-β enhanced tumor immunity mainly through the induction of WT1-specific CTLs, enhancement of NK activity, and promotion of MHC class I expression on the tumor cells. WT1 peptide vaccination combined with IFN-β administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy.

Development of Antigen Specific T Cells After HLA-Mismatched Umbilical Cord Blood Transplantation

Abstract 4544 [Introduction]Hematopoietic cell transplantation (HCT) is a promising strategy for the treatment of both hematologic malignancies and non-malignant hematologic dyscrasias. However, HCT is sometimes complicated by infectious diseases, especially those caused by virus. Although the most important defense mechanism to control viral replication is inducing virus-specific cytotoxic T-lymphocytes (CTLs), the details of CTL development are not fully elucidated in human transplantation. In 1978, Zinkernagel, et al., clarified in murine hematopoietic stem cell transplantation models that MHC-restricted T cells are not governed by donor HLA expressed in bone marrow stem and progenitor cells. Instead, MHC restriction is acquired by interaction with recipient thymic epithelial cells. For this reason, virus-specific T cells are not induced by the mismatched HLA. Due to the increasing number of cord blood and haplo-identical transplantations, many transplantations are being performed under HLA-mismatched conditions. However, the full repercussions of this HLA-mismatch on viral immunity are still unknown. [Clinical case / Methods]For 4 years we followed a 35-year-old patient (HLA-A*24:02, A*33:03) with acute myeloid leukaemia of subtype t(8;21)(q22;q22);RUNX1(AML1)-RUNX1T1(ETO) of the 2008 WHO classification who underwent cord blood (HLA-A*02:01, A*24:02) transplantation following myeloablative conditioning regimen in first complete molecular remission. We analyzed the cytomegalovirus-specific CD8(+) T cells (CMV-CTLs) through HLA-A*02:01 tetramer (HLA-mismatched) and HLA-A*24:02 tetramer (HLA-matched) by flow cytometer. T cell receptor repertoire of these tetramer-positive CMV-CTLs were analyzed by CTL cloning and CDR3 sequencing. [Results / Discussion]The patient had a short period of CMV antigenemia but not CMV disease which was well-controlled by drip infusion of the anti-viral reagent ganciclovir after transplantation. On day 41 after transplantation, only A24-restricted (HLA-matched) tetramer-positive CMV-CTLs were present. By day 195, however, A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs also appeared. We considered that the donor-derived dendritic cells (DCs) induced A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs from naïve T cells, which are originally contained in cord blood inoculum. These mismatched donor HLA restricted memory T cells existed for a long time with a wide-ranging repertoire after transplantation, however, they were considered to have a limited functional role in infection immunity because lungs and intestinal tracts epithelial cells do not express HLA-A2. Usually, cord blood with 0, 1, or 2 loci mismatches is applicable for transplantation. Since HLA-A and B loci are checked only on a serological level, there are some possibilities in which every HLA-A and B loci are mismatched in the genetic level. In such a case, induction of virus-specific CTLs from donor naïve T cells might be impaired and result in uncontrollable viral infections. The findings of this case will be helpful to understand the impact of HLA-mismatch on viral immunity. Disclosures:No relevant conflicts of interest to declare.

How to connect an IgE-driven response with CTL activity?

One of the goals of cell-based immune therapy in cancer is the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses. To achieve this objective, the ability of dendritic cells (DC) to cross-present tumor antigens can be exploited. One of the most efficient pathways for the induction of CTLs by cross-presentation is mediated by immunoglobulins of the IgG class, which are used by DCs to sample antigen in the form of immune complexes via Fc-gamma receptors. Could DCs use an IgE-mediated cross-presentation mechanism in a comparable manner to induce CTLs? We here discuss the potential of two human IgE Fc receptors, FcεRI and FcεRII, to serve as antigen uptake receptors for IgE-mediated cross-presentation. We conclude that the existence of an IgE-mediated cross-presentation pathway would provide a direct link between IgE-driven immune responses and CTL activity.

Immunostimulatory CpG-DNA and PSA-peptide vaccination elicits profound cytotoxic T cell responses

ObjectiveNovel strategies for the treatment of advanced prostate cancer (CaP), including immunotherapy or gene therapy, are currently under evaluation with Sipuleucel-T as first FDA-approved immunotherapeutic. Here, we examine cytosine-phosphorothioate-guanine (CpG)-DNA oligonucleotides (ODN) to boost cytokine responses and costimulatory molecule expression on murine bone marrow-derived dendritic cells (mBMDC). Furthermore, we evaluate the potency of a PSA-peptide based vaccine in combination with CpG-DNA to elicit specific cytotoxic T cell (CTL) responses. Materials and methodsmBMDC were stimulated with CpG-DNA (1668: 5′-TCCATGACGTTCCTGATGCT-3′) or non-stimulatory control-ODN (1720: 5′-TCCATGAGCTTCCTGATGCT-3′). Subsequently, expression of the costimulatory molecules CD40 and CD86 and induction of proinflammatory cytokines (interleukin (IL)-6 and IL-12) were analyzed. For induction of PSA-peptide specific CTL, female C57BL/6 mice were immunized with PSA-peptide 65–73 (HCIRNKSVI) alone or in combination with 1668 or 1720-ODN. In vivo cytotoxicity assay determined PSA-peptide specific cytotoxicity 1 week after vaccination. ResultsTreatment of mBMDC with stimulatory CpG-DNA ODN resulted in pronounced up-regulation of costimulatory molecule expression on mBMDC in a dose-dependent manner. CpG-ODN significantly increased production of IL-6 and IL-12 in mBMDC (P < 0.001). Induction of PSA-peptide specific CTL responses in mice immunized with PSA-peptide and CpG-DNA were significantly greater than those of PSA-peptide and control-ODN immunized mice or PSA-peptide only vaccination. ConclusionsCpG-DNA acts as potent adjuvant for vaccination therapies and elicits profound PSA-peptide specific CTL responses in combination with an immunodominant PSA-peptide. CpG-ODN mediated immunotherapy represents a potentially inexpensive, safe, easy-to-produce, and easy-to-handle treatment alternative. Therefore, further evaluation of CpG-DNA in immunization therapies against CaP is warranted.

The study on the induction of specific immune cytotoxic T lymphocyte responses against pancreatic cancer by transfected dendritic cells with common tumor antigen survivin mRNAs in vitro

Objective To investigate the induction of specific anti-tumor immune response by transfected dendritic cells (DCs) with survivin mRNA of human pancreatic cancer, and to provide the experimental evidences for the treatment of human pancreatic cancer with DCs vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs). After being transcripted and amplified, survivin mRNA was transfected into DCs by electroporation. The expression of survivin in DCs at different time points was detected by quantitative real-time PCR. The survival rate of DCs before and after transfection was determined by MTT method. The induction of specific cytotoxic T lymphocyte (CTL) response by survivin mRNA transfected DCs was measured by 51Cr standard cytotoxicity test. The induction of specific CTL activation by survivin mRNA transfected DCs was evaluated through testing released IFN-γ by ELISA method. Results After survivin mRNA transfection for 48h, the expression of survivin mRNA in DCs reached the highest point (46.09±6.57). After transfection, the survival rate of DCs was stabilized around 80%. The DCs transfected with survivin mRNA could effectively induce HLA-A2+ / survivin+ specific CTL immune responses. Stimulated with pancreatic cancer cell line Capan-2 cells or SCL-1 cells as control group, the IFN-γ released in 24 hours by survivin specific CTL were (28.79±5.70) U/ml and (25.12±2.13) U/ml respectively, there was no significant difference (P=0.761). Conclusion The induction of CTLs by DCs transfected with human pancreatic cancer survivin mRNA could produce specific anti-tumor immunity. Key words: Dendritic cells; Microtubule-associated proteins; RNA, messenger; Transfection; Pancreatic neoplasms; T-lymphocytes, cytotoxic

New Strategy of Adult T-cell Leukemia Treatment Targeted for Anti-tumor Immunity and a Longevity Gene-encoded Protein

Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm with a poor prognosis, developing after long-term infection with human T-cell leukemia virus-1 (HTLV-1). Multiple factors (e.g., virus, host cells, epigenetic aberrations, and immune factors) have been implicated in the development of ATL, although the underlying mechanisms of leukemogenesis have not been fully elucidated. Despite recent progress in both chemotherapy and supportive care for hematological malignancies, the prognosis of ATL is still poor; overall survival at 3 years is only 24%. New strategies for the therapy and prophylaxis of ATL (e.g., vaccines and novel molecular target agents) are still required. This article reviews new strategy of ATL treatment targeted for HTLV-1-specific cytotoxic T-lymphocytes (CTLs) and SIRT1, a longevity gene-encoded protein. HTLV-1-specific CTLs play a critical role in the host immune response against HTLV-1. We have described here the decreased frequency and function of HTLV-1-specific CD8+ T cells in ATL patients and the efficient induction of the HTLV-1-specific CTLs response in human leukocyte antigen-A* 0201-transgenic mice by the HTLV-1/hepatitis B core chimeric particle and oligomannose-coated liposomes encapsulating HTLV-1 epitope without adjuvant, suggesting that the efficient antigen delivery system and CTL induction can be exploited to develop a prophylactic vaccine model against tumors and infectious diseases. Furthermore, our studies suggest that SIRT1, a longevity gene-encoded protein, is a crucial anti-apoptotic molecule in ATL cells, and that SIRT1 inhibitors may be useful therapeutic agents for leukemia, especially in patients with ATL. These studies targeted for anti-tumor immunity such as vaccine and SIRT1 may support the new prophylactic and therapeutic approach for ATL.