Therapeutic EBV-specific T cell cytotoxicity against allogeneic LCLs in vitro directly correlates to intracellular IFN/TNF expression in response to EBV peptide stimulation

Background \u0026 Aim SNBTS has been involved in the manufacture of Epstein-Barr virus-specific T cells (EBV-VST) in treatment of post-transplant lymphroliferative disorder (PTLD) for over 20 years. Manufacture of cells for clinical use relies on comprehensive QC analysis to assess product safety, dose, identity, functionality and stability. We have developed an in vitro cytotoxicity assay of EBV-VST against EBV-infected lymphoblastoid cell line (LCL) targets to measure specific lysis compared to controls. Since the LCLs required for this assay may contain live virus, in the advancing regulatory setting this assay is not easily transferable to an accredited QC lab. The aim of this study was to investigate if VST cytotoxicity directly correlated to any other cytotoxic T cell functional assays such as cytokine production or expression of degranulation markers. Methods, Results \u0026 Conclusion Cryopreserved EBV-VST products were thawed, counted, and split for use in both a cytotoxicity assay and intracellular cytokine/ degranulation assay. For the cytotoxicity assay, HLA-matched and unmatched LCLs were labeled with PKH67, and then co-cultured alongside EBV-VSTs at increasing VST to LCL ratios against suitable controls. Plates were incubated for 5 hours, and cells subsequently labeled with Annexin V and DRAQ7 to assess target apoptotis and necrosis by flow cytometry. For the intracellular assay, EBV-VSTs were stimulated for 5 hours with EBV consensus peptides and then labeled with surface and intracellular markers in the following flow cytomeric panel: CD8, CD4, CD45RO, CD107a, Perforin, Granzyme B, IFNγ, TNFα, IL-2 and DRAQ7. Both assays were performed from the same start material at same time to ensure a direct comparison. The percentage of IFNγ/TNFα co-expressing cells in response to EBV peptide stimulation positively correlated with specific lysis of HLA-matched target LCLs in EBV-VST co-cultures (R2= 0.5602, p=0.008, n=12). No correlation of LCL specific lysis was seen with any of the other degranulation markers, and interestingly no correlation seen with other product-variable parameters (VST viability, CD8/CD4 ratio, number of HLA matches) suggesting these have less direct impact on overall killing efficiency. Flow cytometric analysis of IFNγ/TNFα expression in response to EBV peptide stimulation is an accurate surrogate marker of EBV-infected LCL killing by VSTs, and therefore presents a valid, safe and compliant alternative functional characterisation method to the conventional LCL cytotoxicity assay.