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Updated: Jul 5, 2026
Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis
Published on: November 17, 2020
CD16+ gammadelta T cells mediate antibody dependent cellular cytotoxicity: potential mechanism in the pathogenesis of
Zhihong Chen1, Mark S Freedman
1Department of Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.
Our overall objective is to understand the role of gammadelta T cells in the pathogenesis of the central nervous system (CNS) autoimmune disease multiple sclerosis (MS). We have demonstrated that gammadelta T cells are directly cytotoxic to CNS cells in vitro. Although the exact mechanism of damage in MS is unknown, recent evidence suggests a role for B cells and antibodies to myelin. We were therefore interested in examining whether gammadelta T cells can injure CNS cells via an indirect mechanism involving antibody dependent cellular cytotoxicity. To study this we developed an in vitro flow cytometric cellular cytotoxicity assay (called "FC(3)A") to quantitate the amount of cytotoxicity. We utilized known target cells (Burkitt's B lymphoma) that express CD20, together with a monoclonal antibody (mAb) to CD20, rituximab, that is being studied as a potential treatment for MS. Target cells are first coated with rituximab followed by co-culture with gammadelta T cells derived from patients with MS. Specific lysis of target cells was determined by quantitation of 7-AAD (which increases only upon nuclear disruption indicating cell death). We determined that this lysis was due to gammadelta T cells that express CD16 (Fc gamma receptor) and were therefore capable of binding the rituximab and mediating cytolysis via ADCC. This specific cell lysis correlated with rituximab concentration, E:T ratio, and the surface expression of CD16 on gammadelta T cells. These findings provide a new perspective with regards to the role of gammadelta T cells in the immunopathogenesis of MS and an insight into one of the potential therapeutic effects of rituximab in the treatment of MS. In addition, this new FC(3)A method we developed could readily be adapted to study other types of immune cells suspected of ADCC-type killing.
Our overall objective is to understand the role of gammadelta T cells in the pathogenesis of the central nervous system (CNS) autoimmune disease multiple sclerosis (MS). We have demonstrated that gammadelta T cells are directly cytotoxic to CNS cells in vitro. Although the exact mechanism of damage in MS is unknown, recent evidence suggests a role for B cells and antibodies to myelin. We were therefore interested in examining whether gammadelta T cells can injure CNS cells via an indirect mechanism involving antibody dependent cellular cytotoxicity. To study this we developed an in vitro flow cytometric cellular cytotoxicity assay (called "FC(3)A") to quantitate the amount of cytotoxicity. We utilized known target cells (Burkitt's B lymphoma) that express CD20, together with a monoclonal antibody (mAb) to CD20, rituximab, that is being studied as a potential treatment for MS. Target cells are first coated with rituximab followed by co-culture with gammadelta T cells derived from patients with MS. Specific lysis of target cells was determined by quantitation of 7-AAD (which increases only upon nuclear disruption indicating cell death). We determined that this lysis was due to gammadelta T cells that express CD16 (Fc gamma receptor) and were therefore capable of binding the rituximab and mediating cytolysis via ADCC. This specific cell lysis correlated with rituximab concentration, E:T ratio, and the surface expression of CD16 on gammadelta T cells. These findings provide a new perspective with regards to the role of gammadelta T cells in the immunopathogenesis of MS and an insight into one of the potential therapeutic effects of rituximab in the treatment of MS. In addition, this new FC(3)A method we developed could readily be adapted to study other types of immune cells suspected of ADCC-type killing.
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