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J Immunol Methods. 2012 Jan 31;375(1-2):252-7. doi: 10.1016/j.jim.2011.09.008. Epub 2011 Oct 5.

Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells.

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Vaccine and Gene Therapy Institute - Florida, Port Saint Lucie, FL 34987, USA.


The major function of CD8 T cells is to kill specifically target cells. Moreover in certain incurable diseases, antigen-specific human CD8 T cells are impaired, and assessment of their cytolytic activity could bring insights into their physiopathological role and ways to restore immune dysfunctions for immunotherapeutic purposes. Despite this, T cell cytolytic function has been seldom analyzed thoroughly in humans, due to the lack of approaches well suited for ex vivo assessment of T cell cytotoxicity. Current techniques require prior in vitro expansion of antigen-specific CD8 T cell populations and the use of immortalized cells as targets to measure the cell-mediated killing. Furthermore, bulk cytotoxic activity is frequently measured using percentage of specific lysis calculations that do not quantify actual target cell death and effector numbers at the single cell level. Here we established a new flow cytometry-based assay that allows accurate single-cell analysis of cytotoxic capacity of primary antigen-specific CD8 T cells generated in vivo in humans after antigenic exposure without in vitro amplification that can be used for specificities restricted by different HLAs as target cells are autologous cells. We show that this assay is robust, highly sensitive irrespective of the frequency of antigen-specific CD8 T cells, and allows accurate calculation of the index of cytotoxic capacity in lytic units. This new assay provides a sensitive method to measure the intrinsic cytotoxic activity of antigen-specific CD8 T cells directly ex vivo on human primary cells.

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