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Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications.

ABSTRACT: Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-gamma or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-gamma-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-gamma and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-gamma-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-gamma selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications.

SUBMITTER: Han S

PROVIDER: S-EPMC2729746 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications.

Han Shuhong S Huang Yuju Y Liang Yin Y Ho Yuchin Y Wang Yichen Y Chang Lung-Ji LJ

Journal of hematology & oncology 20090806

Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated ...[more]

PMID: 19660111

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Project description:BackgroundEndoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodal treatment methods that utilize and combine conventional cancer therapies with antigen-specific immunotherapies have emerged as promising approaches for the treatment and control of cancer. However, it is not well known whether endoplasmic reticulum stress-inducing agents can influence the efficacy of tumor antigen-targeting vaccines.MethodsIn the past, we developed a therapeutic human papillomavirus (HPV) DNA vaccine that encodes for calreticulin (CRT) linked to the HPV16 E7 antigen (CRT/E7). In this study, we utilize the CRT/E7 and further encode for an endoplasmic reticulum (ER) stress-inducing agent, 3-bromopyruvate (3-BrPA), in a preclinical model, by harnessing its potential to enhance HPV16 E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors (TC-1 cells). E7-specific CD8+ T cells were added to evaluate the cytotoxicity of luciferase-expressing TC-1 tumor cells treated with 3-BrPA in vitro, as measured with an IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-BrPA-treated TC-1 cells. TC-1 tumor-bearing mice were treated with either 3-BrPA (10 mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30 μg/mouse).ResultsTreatment of E7-expressing TC-1 tumor cells with 3-BrPA induced significantly higher in vitro cytotoxicity and resulted in upregulation of endoplasmic reticulum stress markers (CHOP and GRP78). More importantly, combination treatment of 3-BrPA and the CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice.ConclusionsOur data indicate that 3-BrPA can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation and provide novel strategies for the treatment of HPV-associated diseases.

Project description:Within the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7-H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.

Dataset Information

Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications.

Publications

Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications.

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