Abstract 4121

Reactivation or infection with cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (Ad) and BK virus (BK) are frequent potentially lethal viral complications encountered after allogeneic SCT. Antiviral chemotherapy is not universally effective and carries its own complications. Though adoptive therapy with viral specific T-cells has proven safe and effective for both prevention and treatment of post-transplant viral reactivation, improved strategies are still needed to increase the general applicability of this approach. In this study we tested a simplified rapid strategy of the generation of a single product of CD4+ and CD8+ cytotoxic T-lymphocytes (CTL) lines recognizing CMV, EBV, Ad, BK. We used dendritic cells (DC) derived from elutriated monocytes. Matured DCs were pulsed with overlapping 15mer peptide libraries (pepmixes) spanning CMV-pp65 and IE-1; EBV-LMP2, BZLF1 and EBNA1; Adv-Penton, Hexon; BKV- LT, ST and VP1 alone or in combination (Mix), washed and irradiated. They were subsequently co-cultured with autologous lymphocytes obtained from the initial elutriation in presence of IL-7, IL-15, and low dose IL-2. Reactivity of T cell cultures was analyzed by flow cytometry after one and two rounds of antigenic stimulation and day 9 to day 15 expansion, respectively. For detection of antigen specific CTLs, cells were stimulated with test pepmix or control superantigen (SEB) in the presence of Brefeldin A, anti-CD28 and CD49d for 6 hours, washed, stained for T-cell subset markers and then permeabilized and intracellular-stained for IFNγ, TNFα, IL-2, CD107a. Quadrivirus-specific IFN-γ-producing CD4+ and CD8+ CTLs were successfully induced in all 12 donors (CD4+ 5.8±6.8%, CD8+ 7.3±7.9%). Induction of reactivity in cultures containing individual pepmixes was comparable with results from the cultures containing combination of all studied peptide libraries, supporting the feasibility of developing of a quadrivirus-specific product in a single mixed culture. Two rounds of expansion resulted in significant increase in frequency of CD8+ antigen-specific cells, while there was no change in reactivity of CD4+ cells. The predominant specificity was against CMV in CMV-seropositive donors (n=6; CD3+:8.7±8%, CD4+:3.6±5.9%, CD8+:5.1±3.6%). Significant CMV-reactivity of CTLs was also induced in CMV-seronegative donors (n=6; CD3+:1.6±1%, CD4+:1.2±0.7%, CD8+:0.4±0.3%) and could be increased by performing a second round of antigenic stimulation. The most immunogenic peptides were derived from pp65 antigen for CMV, from BLFZ1 for EBV, from LT for BK and from Hexon for Adv. The expanded T cells retained predominantly naïve or central memory phenotype and only a small subset demonstrated undesirable markers of terminal differentiation or senescence. Furthermore, the anti-viral cells were polyfunctional, as evidenced by simultaneous production with IFNg of TNF-α and (to a lesser degree) IL-2. Thus, we generated CTLs not only capable of specific recognition of viral antigens, but also displaying functional and phenotypic characteristics predictive of high capacity to persist, expand and function after adoptive cell transfer in vivo. In conclusion, we demonstrate a simple, rapid (9 days) GMP-compatible methodology to generate a single preparation of polyclonal CTLs specific for four viruses that are frequent causes of post-transplant mortality or morbidity.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.