title: Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system.
abstract: The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems. We have developed a microraft array methodology that automatically measures the ability of individual T cells to kill a population of target cells and viably sorts specific cells into a 96-well plate for expansion. A human T cell culture was generated against the influenza M1p antigen. Individual microrafts on a 70 × 70 array were loaded with on average 1 CD8+cell from the culture and a population of M1p presenting target cells. Target cell killing, measured by fluorescence microscopy, was quantified in each microraft. The rates of target cell death among the individual CD8+T cells varied greatly; however, individual T cells maintained their rates of cytotoxicity throughout the time course of the experiment enabling rapid identification of highly cytotoxic CD8+T cells. Microrafts with highly active CD8+T cells were individually transferred to wells of a 96-well plate, using a needle-release device coupled to the microscope. Three sorted T cells clonally expanded. All of these expressed high-avidity T cell receptors for M1p/HLA*02:01 tetramers, and 2 of the 3 receptors were sequenced. While this study investigated single T cell cytotoxicity rates against simple targets with subsequent cell sorting, future studies will involve measuring T cell mediated cytotoxicity in more complex cellular environments, enlarging the arrays to identify very rare antigen specific T cells, and measuring single cell CD4+and CD8+T cell proliferation.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill NC and North Carolina State University, Raleigh NC, USA and North Carolina State University, Raleigh NC, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Attayek
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Hunsucker
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA and Department of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill NC and North Carolina State University, Raleigh NC, USA and North Carolina State University, Raleigh NC, USA and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA and Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA and Department of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Armistead
mesh_terms: D000956:Antigens, Viral / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002469:Cell Separation; D002478:Cells, Cultured; D018604:Epitope Mapping / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D007694:Killer Cells, Natural / Q000276:immunology; D056656:Lab-On-A-Chip Devices; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D046888:Tissue Array Analysis / Q000295:instrumentation
chemical_list: D000956:Antigens, Viral