Journal Articles

Innovative Methods

Methods with focus on improving cancer immunotherapy approaches

Gastric cancer vaccines synthesized using a TLR7 agonist and their synergistic antitumor effects with 5-fluorouracil

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BACKGROUND: Vaccines play increasingly important roles in cancer treatment due to their advantages of effective targeting and few side effects. Our laboratory has attempted to construct vaccines by conjugating TLR7 agonists with tumor-associated antigens. Furthermore, immunochemotherapy has recently become an appealing approach to cancer therapy. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, can reportedly potently and selectively kill tumor-associated MDSCs in vivo. METHODS: Gastric cancer vaccines were synthesized by the covalent attachment of our TLR7 agonist with the gastric cancer antigen MG7-Ag tetra-epitope, leading to T7 - ML (linear tetra-epitope) and T7 - MB (branched tetra-epitope). Cytokines induced by the vaccines in vitro were assessed by ELISA. A tumor challenge model was created by treating BALB/c mice on either a prophylactic or therapeutic vaccination schedule. 5-FU was simultaneously applied to mice in the combination treatment group. CTL and ADCC activities were determined by the LDH method, while CD3(+)/CD8(+), CD3(+)/CD4(+) T cells and MDSCs were evaluated by flow cytometry. RESULTS: In vitro, rapid TNF-alpha and IL-12 inductions occurred in BMDCs treated with the vaccines. In vivo, among all the vaccines tested, T7 - MB most effectively reduced EAC tumor burdens and induced CTLs, antibodies and ADCC activity in BALB/c mice. Immunization with T7 - MB in combination with 5-FU chemotherapy reduced tumor sizes and extended long-term survival rates, mainly by improving T cell responses, including CTLs, CD3(+)/CD8(+) and CD3(+)/CD4(+) T cells. 5-FU also enhanced the T7 - MB efficiency by reversing immunosuppressive factors, i.e., MDSCs, which could not be validly inhibited by the vaccines alone. In addition, T7 - MB repressed tumor growth and immune tolerance when the therapeutic schedule was used, although the effects were weaker than those achieved with either T7 - MB alone or in combination with 5-FU on the prophylactic schedule. CONCLUSIONS: A novel effective gastric cancer vaccine was constructed, and the importance of branched multiple antigen peptides and chemical conjugation to vaccine design were confirmed. The synergistic effects and mechanisms of T7 - MB and 5-FU were also established, observing mainly T cell activation and MDSC inhibition.

Author Info: (1) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. (2) The 3rd Affiliated Hospital of Shenzhen University, Shenzhen, 518001, Guangdong

Author Info: (1) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. (2) The 3rd Affiliated Hospital of Shenzhen University, Shenzhen, 518001, Guangdong, China. (3) Department of Biology and School of Medicine, Southern University of Science and Technology, Shenzhen, 518055, Guangdong, China. (4) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. (5) The 3rd Affiliated Hospital of Shenzhen University, Shenzhen, 518001, Guangdong, China. (6) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. (7) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. (8) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. (9) School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. gyjin@szu.edu.cn. Cancer Research Center, Shenzhen University Health Science Center, Shenzhen, 518060, Guangdong, China. gyjin@szu.edu.cn.

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Cytomegalovirus: an unlikely ally in the fight against blood cancers

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CMV infection is a potentially fatal complication in patients receiving HSCT, but recent evidence indicates that CMV has strong anti-leukemia effects due in part to shifts in the composition of NK-cell subsets. NK-cells are the primary mediators of the anti-leukemia effect of allogeneic HSCT and infusion of allogeneic NK-cells has shown promise as a means of inducing remission and preventing relapse of several different hematologic malignancies. The effectiveness of these treatments is limited, however, when tumors express HLA-E, a ligand for the inhibitory receptor NKG2A which is expressed by the vast majority of post-transplant reconstituted and ex vivo expanded NK-cells. It is possible to enhance NK-cell cytotoxicity against HLA-E(pos) malignancies by increasing the proportion of NK-cells expressing NKG2C (the activating receptor for HLA-E) and lacking the corresponding inhibitory receptor NKG2A. The proportion of NKG2C(pos) /NKG2A(neg) NK-cells is typically low in healthy adults, but it can be increased by CMV infection or ex vivo expansion of NK-cells using HLA-E transfected feeder cells and IL-15. In this review, we will discuss the role of CMV-driven NKG2C(pos) /NKG2A(neg) NK-cell expansion on anti-tumor cytotoxicity and disease progression in the context of hematologic malignancies, and explore the possibility of harnessing NKG2C(pos) /NKG2A(neg) NK-cells for cancer immunotherapy. This article is protected by copyright. All rights reserved.

Author Info: (1) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences

Author Info: (1) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences, The University of Arizona, 1177 E. Fourth Street, Tucson, Arizona, 85721, USA. (2) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences, The University of Arizona, 1177 E. Fourth Street, Tucson, Arizona, 85721, USA. (3) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences, The University of Arizona, 1177 E. Fourth Street, Tucson, Arizona, 85721, USA.

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VISTA expression on tumor-infiltrating inflammatory cells in primary cutaneous melanoma correlates with poor disease-specific survival

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Adaptive immune responses contribute to the pathogenesis of melanoma by facilitating immune evasion. V-domain Ig suppressor of T-cell activation (VISTA) is a potent negative regulator of T-cell function and is expressed at high levels on monocytes, granulocytes, and macrophages, and at lower densities on T-cell populations within the tumor microenvironment. In this study, 85 primary melanoma specimens were selected from pathology tissue archives and immunohistochemically stained for CD3, PD-1, PD-L1, and VISTA. Pearson's correlation coefficients identified associations in expression between VISTA and myeloid infiltrate (r = 0.28, p = 0.009) and the density of PD-1+ inflammatory cells (r = 0.31, p = 0.005). The presence of VISTA was associated with a significantly worse disease-specific survival in univariate analysis (hazard ratio = 3.57, p = 0.005) and multivariate analysis (hazard ratio = 3.02, p = 0.02). Our findings show that VISTA expression is an independent negative prognostic factor in primary cutaneous melanoma and suggests its potential as an adjuvant immunotherapeutic intervention in the future.

Author Info: (1) Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Department of Medicine, Santa Barbara Cottage Hospital, Santa Barbara, CA, USA. (2) Department of Pathology

Author Info: (1) Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Department of Medicine, Santa Barbara Cottage Hospital, Santa Barbara, CA, USA. (2) Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA. (3) Biostatistics Shared Resource, Norris Cotton Cancer Center, Dartmouth-Hitchock Medical Center, Lebanon, NH, USA. (4) Department of Medicine, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA. (5) Departments of Biomedical Data Sciences, Molecular and Systems Biology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, USA. (6) Department of Microbiology and Immunology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. (7) Department of Surgery, Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA. (8) Department of Microbiology and Immunology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. (9) Roswell Park Cancer Institute, University of Buffalo, The State University of New York, Elm and Carlton, Buffalo, NY, 14263, USA. marc.ernstoff@roswellpark.org.

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Induction of necrotic cell death and activation of STING in the tumor microenvironment via cationic silica nanoparticles leading to enhanced antitumor immunity

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Nanotechnology has demonstrated tremendous clinical utility, with potential applications in cancer immunotherapy. Although nanoparticles with intrinsic cytotoxicity are often considered unsuitable for clinical applications, such toxicity may be harnessed in the fight against cancer. Nanoparticle-associated toxicity can induce acute necrotic cell death, releasing tumor-associated antigens which may be captured by antigen-presenting cells to initiate or amplify tumor immunity. To test this hypothesis, cytotoxic cationic silica nanoparticles (CSiNPs) were directly administered into B16F10 melanoma implanted in C57BL/6 mice. CSiNPs caused plasma membrane rupture and oxidative stress of tumor cells, inducing local inflammation, tumor cell death and the release of tumor-associated antigens. The CSiNPs were further complexed with bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a molecular adjuvant which activates the stimulator of interferon genes (STING) in antigen-presenting cells. Compared with unformulated c-di-GMP, the delivery of c-di-GMP with CSiNPs markedly prolonged its local retention within the tumor microenvironment and activated tumor-infiltrating antigen-presenting cells. The combination of CSiNPs and a STING agonist showed dramatically increased expansion of antigen-specific CD8+ T cells, and potent tumor growth inhibition in murine melanoma. These results demonstrate that cationic nanoparticles can be used as an effective in situ vaccine platform which simultaneously causes tumor destruction and immune activation.

Author Info: (1) Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, USA. haipengl.liu@wayne.edu. (2) Department of Chemical Engineering and Materials Science, Wayne

Author Info: (1) Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, USA. haipengl.liu@wayne.edu. (2) Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, USA. haipengl.liu@wayne.edu. (3) Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, USA. haipengl.liu@wayne.edu. (4) Department of Oncology, Wayne State University, Detroit, Michigan 48201, USA and Tumor Biology and Microenvironment Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA. (5) Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, USA. haipengl.liu@wayne.edu. (6) Department of Oncology, Wayne State University, Detroit, Michigan 48201, USA and Tumor Biology and Microenvironment Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA. (7) Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, USA. haipengl.liu@wayne.edu and Department of Oncology, Wayne State University, Detroit, Michigan 48201, USA and Tumor Biology and Microenvironment Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA.

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Immune Checkpoint Inhibitors in the Treatment of Patients with Neuroendocrine Neoplasia

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BACKGROUND: Well-differentiated neuroendocrine neoplasms (NENs) are usually controlled by antiproliferative, local ablative and/or radionuclide therapies, whereas poorly differentiated NENs generally require cytotoxic chemotherapy. However, treatment options for patients with advanced/metastatic high-grade NENs remain limited. METHOD: Review of the literature and international congress abstracts on the efficacy and safety of immunotherapy by checkpoint inhibition in advanced/metastatic NENs. RESULTS: Evidence points to an important role of immune phenomena in the pathogenesis and treatment of neuroendocrine tumors (NETs). Programmed cell death 1 (PD-1) protein and its ligand are mainly expressed in poorly differentiated NENs. Microsatellite instability and high mutational load are more pronounced in high-grade NENs and may predict response to immunotherapy. Clinical experience of immune checkpoint blockade mainly exists for Merkel cell carcinoma, a high-grade cutaneous neuroendocrine carcinoma (NEC), which has led to approval of the anti-PD-1 antibody avelumab. In addition, there is anecdotal evidence for the efficacy of checkpoint inhibitors in large-cell lung NECs, ovarian NECs and others, including gastroenteropancreatic NENs. Currently, phase II studies investigate PDR001, pembrolizumab, combined durvalumab and tremelimumab, and avelumab treatment in patients with advanced/metastatic NENs. CONCLUSION: Immune checkpoint inhibitors are a promising therapeutic option, especially in progressive NECs or high-grade NETs with high tumor burden, microsatellite instability, and/or mutational load.

Author Info: (1) (2)

Author Info: (1) (2)

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Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function

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When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8(+) T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8(+) T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8(+) T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8(+) T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8(+) T cell activation, mediated by the binding of empty HLA-I to CD8.

Author Info: (1) Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States. (2) Department of Microbiology and Immunology, Yerkes National Primate Research Center, Emory

Author Info: (1) Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States. (2) Department of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, United States. (3) Research and Development, Sirona Genomics, Immucor, Inc, Mountain View, United States. (4) Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States.

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A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity

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The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1alpha). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo-like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

Author Info: (1) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (2) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (3) Life Sciences Division, Corning

Author Info: (1) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (2) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (3) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States.

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Mycobacterium tuberculosis PPE60 antigen drives Th1/Th17 responses via Toll-like receptor 2-dependent maturation of dendritic cells

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Targeting of Mycobacterium tuberculosis (MTB) PE/PPE antigens that induce type 1 helper T cell (Th1) and Th17 responses represents a crucial strategy for the development of tuberculosis (TB) vaccines. However, only few PE/PPE antigens induce these responses. Here, we sought to determine how the cell wall-associated antigen PPE60 (Rv3478) activates dendritic cell (DC) maturation and T-cell differentiation. We observed that PPE60 induces DC maturation by augmenting the protein expression of cluster of differentiation 80 (CD80) and CD86, and major histocompatibility complex (MHC) class I and MHC class II on the cell surface. PPE60 also stimulated the production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, IL-12p70, and IL-23p19, but not IL-10. This induction was mediated by Toll-like receptor 2 (TLR2) and followed by activation of p38, c-Jun N-terminal kinase (JNK), and NF-kappaB signaling. PPE60 enhanced MHC-II expression and promoted antigen processing by DCs in a TLR2-dependent manner. Moreover, PPE60-stimulated DCs directed naive CD4(+) T cells to produce IFN-gamma, IL-2, and IL-17A, expanding the Th1 and Th17 responses, along with activation of T-Bet and RAR-related orphan receptor C (RORgammat), but not GATA-3. Moreover, PPE60 activated the NLRP3 inflammasome, followed by caspase-1-dependent IL-1beta and IL-18 synthesis in DCs. Of note, pharmacological inhibition of NLRP3 activation specifically attenuated IFN-gamma and IL-17A secretion into the supernatant from CD4(+) T cells co-cultured with PPE60-activated DCs. These findings indicate that PPE60 induces Th1 and Th17 immune responses by activating DCs in a TLR2-dependent manner, suggesting PPE60's potential for use in MTB vaccine development.

Author Info: (1) GMU-GIBH Joint School of Life Science, Guangzhou Medical University, China. (2) Guangdong Second Provincial General Hospital, China. (3) GMU-GIBH Joint School of Life Science

Author Info: (1) GMU-GIBH Joint School of Life Science, Guangzhou Medical University, China. (2) Guangdong Second Provincial General Hospital, China. (3) GMU-GIBH Joint School of Life Science, Guangzhou Medical University, China. (4) GMU-GIBH Joint School of Life Science, Guangzhou Medical University, China. (5) State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, China. (6) State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, China. (7) Department of Laboratory Medicine and Central Laboratories, Guangdong Second Provincial General Hospital, China. (8) State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, China.

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The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

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Cytotoxic necrotizing factor 1 (CNF1) is a bacterial protein toxin primarily expressed by pathogenic Escherichia coli strains, causing extraintestinal infections. The toxin is believed to enhance the invasiveness of E. coli by modulating the activity of Rho GTPases in host cells, but it has interestingly also been shown to promote inflammation, stimulate host immunity and function as a potent immunoadjuvant. The mechanisms underlying the immunostimulatory properties of CNF1 are, however, poorly characterized, and little is known about the direct effects of the toxin on immune cells. Here, we show that CNF1 induces expression of maturation markers on human immature monocyte-derived dendritic cells (moDCs) without compromising cell viability. Consistent with the phenotypic maturation, CNF1 further triggered secretion of proinflammatory cytokines and increased the capacity of moDCs to stimulate proliferation of allogenic naive CD4+ T cells. A catalytically inactive form of the toxin did not induce moDC maturation, indicating that the enzymatic activity of CNF1 triggers immature moDCs to undergo phenotypic and functional maturation. As the maturation of dendritic cells plays a central role in initiating inflammation and activating the adaptive immune response, the present findings shed new light on the immunostimulatory properties of CNF1 and may explain why the toxin functions as an immunoadjuvant.

Author Info: (1) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. lgmas@sund.ku.dk. (2) Italian Center for Global Health, Istituto Superiore di

Author Info: (1) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. lgmas@sund.ku.dk. (2) Italian Center for Global Health, Istituto Superiore di Sanita; Viale Regina Elena 299, 00161 Rome, Italy. alessia.fabbri@iss.it. (3) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. mnamini@sund.ku.dk. (4) Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. mgivskov@sund.ku.dk. (5) Italian Center for Global Health, Istituto Superiore di Sanita; Viale Regina Elena 299, 00161 Rome, Italy. carla.fiorentini@iss.it. (6) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. thorkr@sund.ku.dk.

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PRAME peptide-specific CD8(+) T cells represent the predominant response against leukemia-associated antigens (LAAs) in healthy individuals

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Antigen-specific T cells isolated from healthy individuals (HIs) have shown great therapeutic potential upon adoptive transfer for the treatment of viremia in immunosuppressed patients. The lack of comprehensive data on the prevalence and characteristics of leukemia associated antigen (LAA)-specific T cells in HIs still limits such an approach for tumor therapy. Therefore, we have investigated T cell responses against prominent candidates comprising WT1, PRAME, Survivin, NY-ESO and p53 by screening PBMCs from HIs using intracellular IFN-gamma staining following provocation with LAA peptide mixes. Here, we found predominantly poly-functional effector/effector memory CCR7(-) /CD45RA(+/-) /CD8(+) LAA peptide-specific T cells with varying CD95 expression in 34 of 100 tested HIs, whereas CD4(+) T cells responses were restricted to 5. Most frequent LAA peptide-specific T cell responses were directed against WT1 and PRAME peptides with a prevalence of 20% and 17%, respectively, showing the highest magnitude (0.16% +/- 0.22% (mean+/-SD)) for PRAME peptides. Cytotoxicity of PRAME peptide-specific T cells was demonstrated by specific killing of PRAME peptide-pulsed T2 cells. Furthermore, the proliferative capacity of PRAME peptide-specific T cells was confined to HIs responsive towards PRAME peptide challenge corroborating the accuracy of the screening results. In conclusion, we identified PRAME as a promising target antigen for adoptive leukemia therapy. This article is protected by copyright. All rights reserved.

Author Info: (1) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden

Author Info: (1) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany. (2) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany. (3) Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. (4) German Cancer Research Center (DKFZ), Heidelberg, Germany. German Cancer Consortium (DKTK), Dresden, Germany. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. Institute of Immunology, Medical Faculty, TU Dresden, Dresden, Germany. National Center for Tumor Diseases, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany. (5) German Cancer Research Center (DKFZ), Heidelberg, Germany. German Cancer Consortium (DKTK), Dresden, Germany. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. Department of Medicine 1, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany. National Center for Tumor Diseases, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany. (6) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany. German Cancer Research Center (DKFZ), Heidelberg, Germany. German Cancer Consortium (DKTK), Dresden, Germany. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany. (7) Experimental Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU Dresden, Germany. Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Dresden, Germany.

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