A novel flow cytometric assay for evaluating cell-mediated cytotoxicity.
Comprehensive evaluation of cell-mediated cytotoxicity is very important, especially in the clinical setting, when a surrogate immunologic endpoint that correlates with a clinical outcome needs to be defined. With the objective of simultaneously evaluating target cell death and effector cell frequency, the authors combined the measuring of the expression of the degranulation marker CD107a by effector cells with the apoptosis marker annexin V binding to target cells. Using human cytotoxic T lymphocytes, the authors found a significant incubation time-dependent increase of surface CD107a expression on effector cells due to degranulation. A parallel increase of annexin V binding to target cells due to target cell apoptosis was also found. These two parameters have shown excellent correlation in all effector/target cell systems studied. To find possible connections between effector cell degranulation (CD107a expression), granzyme B secretion, and target cell death (annexin V binding), the GrB ELISPOT assay and flow cytometric assay were performed and excellent cross-correlation was found between all three parameters. The specificity of the assay was also shown. These data show that this novel assay allows measurement of cytolytic cell activation and frequency as well as target cell death in the same sample.
Laboratory of Cell-Mediated Immunity, Clinical Services Program, SAIC-Frederick, Inc., Frederick, Maryland 21702, USA.
Mark W
MW
Burkett
Kimberly A
KA
Shafer-Weaver
Susan
S
Strobl
Michael
M
Baseler
Anatoli
A
Malyguine
D017304:Annexin A5 / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D053804:Granzymes; D006801:Humans; D008212:Lymphocyte Depletion; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D010446:Peptide Fragments / Q000276:immunology; D012697:Serine Endopeptidases / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology
D017304:Annexin A5; D015703:Antigens, CD; D000939:Epitopes; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D010446:Peptide Fragments; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C448634:influenza matrix peptide (58-66); C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases