Targeted in-vitro-stimulation reveals highly proliferative multi-virus-specific human central memory T cells as candidates for prophylactic T cell therapy

Adoptive T cell therapy (ACT) has become a treatment option for viral reactivations in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Animal models have shown that pathogen-specific central memory T cells (T(CM)) are protective even at low numbers and show long-term survival, extensive proliferation and high plasticity after adoptive transfer. Concomitantly, our own recent clinical data demonstrate that minimal doses of purified (not in-vitro- expanded) human CMV epitope-specific T cells can be sufficient to clear viremia. However, it remains to be determined if human virus-specific T(CM) show the same promising features for ACT as their murine counterparts. Using a peptide specific proliferation assay (PSPA) we studied the human Adenovirus- (AdV), Cytomegalovirus- (CMV) and Epstein-Barr virus- (EBV) specific T(CM) repertoires and determined their functional and proliferative capacities in vitro. T(CM) products were generated from buffy coats, as well as from non-mobilized and mobilized apheresis products either by flow cytometry-based cell sorting or magnetic cell enrichment using reversible Fab-Streptamers. Adjusted to virus serology and human leukocyte antigen (HLA)-typing, donor samples were analyzed with MHC multimer- and intracellular cytokine staining (ICS) before and after PSPA. T(CM) cultures showed strong proliferation of a plethora of functional virus-specific T cells. Using PSPA, we could unveil tiniest virus epitope-specific T(CM) populations, which had remained undetectable in conventional ex-vivo-staining. Furthermore, we could confirm these characteristics for mobilized apheresis- and GMP-grade Fab-Streptamer-purified T(CM) products. Consequently, we conclude that T(CM) bare high potential for prophylactic low-dose ACT. In addition, use of Fab-Streptamer-purified T(CM) allows circumventing regulatory restrictions typically found in conventional ACT product generation. These GMP-compatible T(CM) can now be used as a broad-spectrum antiviral T cell prophylaxis in alloHSCT patients and PSPA is going to be an indispensable tool for advanced T(CM) characterization during concomitant immune monitoring.