Assessment of the cytolytic potential of a multivirus-targeted T cell therapy using a vital dye-based, flow cytometric assay

Reliable and sensitive characterization assays are important determinants of the successful clinical translation of immunotherapies. For the assessment of cytolytic potential, the chromium 51 (⁵¹Cr) release assay has long been considered the gold standard for testing effector cells. However, attaining the approvals to access and use radioactive isotopes is becoming increasingly complex, while technical aspects [i.e. sensitivity, short (4-6 hours) assay duration] may lead to suboptimal performance. This has been the case with our ex vivo expanded, polyclonal (CD4+ and CD8+) multivirus-specific T cell (multiVST) lines, which recognize 5 difficult-to-treat viruses [Adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpes virus 6 (HHV6)] and when administered to allogeneic hematopoietic stem cell (HCT) or solid organ transplant (SOT) recipients have been associated with clinical benefit. However, despite mediating potent antiviral effects <i>in vivo</i>, capturing <i>in vitro</i> cytotoxic potential has proven difficult in a traditional ⁵¹Cr release assay. Now, in addition to cytotoxicity surrogates, including CD107a and Granzyme B, we report on an alternative, vital dye -based, flow cytometric platform in which superior sensitivity and prolonged effector:target co-culture duration enabled the reliable detection of both CD4- and CD8-mediated <i>in vitro</i> cytolytic activity against viral targets without non-specific effects.