Opportunistic viral infections persist as the main cause of post-transplantation morbidity and mortality in allogeneic hematopoietic stem-cell recipients [1], [2], [3], [4], [5], [6]. Even though some clinically problematic viruses can be treated or avoided by antiviral therapies, they are not always effective and at times can lead to undesirable outcomes as significant adverse effects [7], [8], [9], [10], [11].

In contrast, the adoptive transfer of virus-specific T cells (VST) has shown efficacy for the treatment of several viral pathogens [12], [13], [14].

In addition, cryopreserved VST cell lines for diverse viruses, are obtained from healthy donors and cryopreserved for immediate availability and, are being successfully used as a third-party therapy in post-transplant settings [14], [15], [16], [17].

It is difficult to develop or improve a cell therapy product that accomplish all Good Manufacturing Practices (GMP) requirements necessary for clinical grade products. Therefore, in targeting cytomegalovirus (CMV), we developed an innovative approach for generating a highly specific VST product following all GMP requirements [13], [18], [19], [20].

First, we have used, a sterile disposable compartment named the Leukoreduction System Chamber (LRS-chamber) from the apheresis platelet donation kit as starting material. This compartment is intended to reduce leukocytes during the assortment of platelet bags; it is discarded after the procedure is completed; and,it has proven to be very rich in T cells. During platelet donation, whole blood from a donor enters the apheresis equipment and passes through this chamber. In the LSR-chamber, whole blood is separated under centrifugation into componentt layers, while platelets and plasma are located in the lower part of the chamber and are drawn out to the collection bag; in the upper part of the chamber, the layers are initiated with red blood cells, granulocytes, monocytes and lymphocytes that are returned to the donor. Thus, cells from the LRS-chamber are distributed accordingly to the platelet collection bag or returned to the donor. However, a small volume of blood is retained in the chamber, and it is mainly composed of cells located in the chamber’s middle lower layer, such as lymphocytes and monocytes [21], [22].

For expansion, we have developed a protocol based on previous published manuscripts that verified several cytokine combinations for VST [23], [24], [25], [26]. Our protocol for VST expansion uses multiple stimulations with CMV capsid phosphoprotein 65 (pp65) overlapping peptides [24], [25] and, two mitotic cytokines, interleukin (IL)-2 and IL-7, combined. IL-7 has demonstrated to play a role in the maintenance and survival of T lymphocytes in culture [26]. Additionally, our results have shown that the combination of IL-2 and IL-7 stimulate VST proliferation favoring T cell (CD4 and CD8) effector memory profiles.

Furthermore, to diminish the presence of expanded non-VST, this protocol has selected these cells by magnetic selection in a closed automated system after expansion, enhancing the final product purity and specificity. Consequently, we developed a high, pure cytotoxic T cell (CTL) anti-CMV product, that was preclinically tested for specificity in vitro and in vivo for persistence, biodistribution, and toxicity using NOD-scid mice.