A cost-effective strategy for selection of third-party donors for a virus-specific T-cell bank for an Asian patient population.

BACKGROUND AIMS: Third party virus-specific T cells (VST) has shown efficacy for opportunistic virus infection which do not have effective treatment or are drug-refractory. We describe our preparatory work in setting up a third-party VST bank for a multi-ethnic Asian population. METHODS: Discarded white cells from regular blood bank plateletpheresis donors with known locally prevalent HLA antigens were cultured in small scale to generate VST against Adenovirus, BK virus, Cytomegalovirus, Epstein-Barr virus, and Human Herpes Virus 6. Multi-virus specific T cells (multi-VST) were also generated against all 5 viruses in single cultures. A strategy of allelic typing for donors with good and broad-spectrum cytotoxicity together with consideration on HLA restriction for the virus epitope was used to select combinations of VST lines for a hypothetical third party VST bank. The breadth of coverage based on these selection criteria was validated using our database of 100 post haematopoietic stem cell transplant patients. RESULTS: We show that 50%, 42%, 56%, 56% and 42% of single VST cultures demonstrated specific cytotoxicity against AdV, BKV, CMV, EBV and HHV6 respectively. Twenty four of the 36 multi-VST lines showed activity against at least 2 of the 5 viruses studied. A carefully selected combination of just 6 VST lines can offer VST with at least 1 allelic match to 99% of potential recipients, while 92% can find 2 allelic matches and 79% can find 3 allelic matches. CONCLUSIONS: This preparatory work confirms that a cost-effective strategy recruiting a small number of pre-characterized donors can generate VST lines with broad coverage for a multi-ethnic Asian patient population, thereby laying the foundation for setting up of a third party VST bank for Asian patients.

A novel simplified method of generating cytomegalovirus-specific cytokine-induced killer cells of high specificity and superior potency with GMP compliance.

We describe a method of rendering polyclonal cytokine-induced killer cells (CIK) specific against cytomegalovirus (CMV), focusing on GMP compliance. Peripheral blood mononuclear cells (PBMNC) are stimulated with pooled CMV peptides pp65 and IE-1 for 16-24 h and the reactive T cell subset which up-regulate CD137 is further co-stimulated with anti-CD137, followed by expansion in G-Rex flasks under standard CIK culture condition. This method generates a large number CMV-specific CIK with superior potency compared to published method currently in clinical trials. The cytotoxicity as measured by chromium release assay correlates with the upregulation of CD107a upon peptide re-challenge as measured by flow cytometry. CMV-CIK at maturity consist of mainly late effector memory CD8 T cells and have a skewed TCR repertoire with preferential expansion of a few families. Such CMV-CIK retain their function after freezing and thawing. CMV-CIK thus generated is ready for clinical trial against drug-resistant CMV disease.