Ex vivo expansion of immature and mature T cells derived from umbilical cord blood (UCB)

Abstract

Introduction: Unrelated umbilical cord blood (UCB) is an effective source of allogeneic hematopoietic stem cells for transplantation therapy in patients lacking matched bone marrow donors. Opportunistic infection is the major cause of mortality post transplant, due in part to the immunologic naiveté of the UCB T-cells. We hypothesized that UCB derived immature and mature T-cells could be expanded ex-vivo, and utilized a patient-derived skin stromal layer with a supplemental cytokine cocktail to support the growth of bulk UCB cells. The long term goal is to provide UCB-derived adoptive immunotherapy in UCBT recipients. Methods: Patient derived skin fibroblasts were cultured in a media of IMDM, 10% fetal calf serum and 10% horse serum for 14 days, then irradiated to 5,000 cGy. Cryopreserved UCB cells were thawed and cultured on this stromal layer in the same media supplemented with a cytokine cocktail of interleukin-7 (IL-7) (10 ng/ml), flt-3 ligand (10 ng/ml), and stem cell factor (50 ng/ml). UCB cells were cultured with cytokines alone and skin alone as controls. As a 2nd phase, interleukin-2 (IL-2) was added at day 14 for further expansion, with cells cultured for an additional 14 days thereafter. Cell count, viability, and FACs analysis were performed. Results: Gating on CD3+, in cells cultured with both skin stroma and cytokines mean fold increases of 6 in CD4+, 11 in CD8+ and 34 in CD4+/CD8+ cells were seen. With cytokines alone, mean fold increases were 3, 4 and 10 respectively. Minimal expansion occurred with skin alone. With cytokines and IL-2, mean fold increases were 4 in CD4+, 4 in CD8+ and 10 in CD4+/CD8+ cells. Maximal expansion occurred with skin stroma, the cytokine cocktail and IL-2, with mean fold increases of 9 in CD4+, 11 in CD8+ and 53 in CD4+/CD8+ cells. Results are summarized in the table below. Conclusions: The expansion of T-cells from unfractionated, red blood cell depleted, cryopreserved UCB can be accomplished using a cytokine cocktail of IL-7, Flt 3 ligand and SCF over a patient derived, irradiated skin stromal layer. Maximal expansion occurs with the addition of IL-2 and was greatest in the immature CD4+/CD8+ T-cell subset. We hypothesize that this subset of T cells could serve as a target population for adoptive immunotherapy, however further testing will determine the optimal cellular target and conditions for ex vivo expansion and immunization. These cells could then be used to augment immune reconstitution after UCBT.