title: Evaluating antigen-specific cytotoxic T lymphocyte responses by a novel mouse granzyme B ELISPOT assay.
abstract: We have established novel ELISA- and ELISPOT-based assays specific for the detection of a potent cytotoxic mediator, granzyme B (GrB), for the assessment of antigen-specific cytotoxic T lymphocyte responses in mice. The sensitivity and specificity of our assays was demonstrated by ELISA using purified mouse GrB and supernatants and cell lysates of cytotoxic lymphocytes derived from GrB-deficient mice. No reactivity was observed by the GrB ELISA in GrB-deficient cells. The mouse GrB ELISPOT was successfully employed to detect antigen-specific effector cell responses of two CTL clones, producing GrB ELISPOT results that correlated strongly with target cell lysis, as assessed by 51Cr-release. Furthermore, we were able to demonstrate direct correlations between GrB ELISPOT and killing by LCMV gp33-specific effector and memory T cells generated in vivo. Thus, the mouse GrB ELISPOT may be used to detect cytotoxic responses, at the single-cell level, for the functional assessment of antigen-specific CD8+ T cell responses in mouse models of infection.
authors.affiliation: Department of Medical Microbiology and Immunology, Rm 6-60, HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Ewen
authors.forename: Jianhui
authors.initials: J
authors.lastname: Rong
authors.forename: Andy I
authors.initials: AI
authors.lastname: Kokaji
authors.forename: R Chris
authors.initials: RC
authors.lastname: Bleackley
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Kane
mesh_terms: D000818:Animals; D000956:Antigens, Viral; D015550:Cell Degranulation; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods* / Q000706:statistics & numerical data; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005260:Female; D053804:Granzymes; D007156:Immunologic Memory; D066298:In Vitro Techniques; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis* / Q000096:biosynthesis; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology* / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases