Automating procedures for processing, cryopreservation, storage, and manipulation of human peripheral blood mononuclear cells

摘要

Cell-mediated immune responses have been found to play a crucial role in the protection from, or amelioration of, many human diseases. There are many vaccine trials being conducted where the most important immune assessment consists of measuring T cell responses. The most readily accessible source of T cells is the peripheral blood. Thus, collection, processing, cryopreservation, storage, and manipulation of human peripheral blood mononuclear cells (PBMCs) are all key steps for assessment of vaccine and disease-induced immune responses. Automation of these procedures is not currently practiced widely. This article reviews current practices and explores the need for automation. Cell-mediated immune responses have been found to play a crucial role in the protection from, or amelioration of, many human diseases. There are many vaccine trials being conducted where the most important immune assessment consists of measuring T cell responses. The most readily accessible source of T cells is the peripheral blood. Thus, collection, processing, cryopreservation, storage, and manipulation of human peripheral blood mononuclear cells (PBMCs) are all key steps for assessment of vaccine and disease-induced immune responses. Automation of these procedures is not currently practiced widely. This article reviews current practices and explores the need for automation. Introduction: why t cells are so important and why we need functional viable t cells for immunogenicity assessmentsBecause of the human immunodeficiency virus-1 (HIV-1) epidemic, a new era in technology development has been ushered in. New discoveries not only for molecular biology but also for immunology and, in particular, for cellular immunology have occurred since HIV-1 was discovered 20 years ago. From studies in HIV-1 infected individuals, as well as individuals that were exposed to but not infected with HIV-1, it soon became apparent that CD8+ T cells were critical for protection. Thus, designing HIV vaccines and assessing HIV disease required a shift from not just examining antibody responses but, in addition, examining CD8+ T cell responses.1Esser M.T. Marchese R.D. Kierstead L.S. et al.Memory T cells and vaccines.Vaccine. 2003; 21: 419-430Crossref PubMed Scopus (130) Google Scholar,2McMichael A. Rowland-Jones S. Cellular immune responses to HIV.Nature. 2001; 410: 980-986Crossref PubMed Scopus (515) Google Scholar For other infectious diseases, such as malaria and tuberculosis, and cancers, disease is at least partially controlled by T cell immune responses.3Aidoo I. Udhayakumar I. Field studies of cytotoxic T lymphocytes in malaria infections: Implications for malaria vaccine development.Parasitol Today. 2000; 16: 50-56Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 4Kaufmann S.H. Protection against tuberculosis: Cytokines, T cells, and macrophages.Ann. Rheum. Dis. 2002; 61: 54-58Crossref Scopus (161) Google Scholar, 5Rosenberg S.A. Progress in human tumour immunology and immunotherapy.Nature. 2001; 411: 380-384Crossref PubMed Scopus (1172) Google Scholar Thus, an important objective of human clinical trials of HIV, tuberculosis, malaria, and cancer vaccines has become the measurement of vaccine and disease-induced T cell responses. For measuring T cell responses, assays such as the traditional semiquantitative cytotoxic T cell (CTL) and lymphoproliferative assays (LPA) have historically been used. More recently, the tetramer, enzyme-linked immunospot assay (ELISPOT) and intracellular cytokine staining (ICS) assays have been employed.6Hickling, J. K. Measuring T-lymphocyte function. Expert reviews in molecular medicine. http://www.expertreviews.org (accessed Oct 1998).Google Scholar, 7Clay T. Hobeika A. Mosca P. Lyerly H. Morse M. Assays for monitoring cellular immune responses to active immunotherapy of cancer.Clin. Cancer Res. 2001; 7: 1127-1135PubMed Google Scholar, 8Coulie P.G. Bruggen P. T-cell responses of vaccinated cancer patients.Current Opini