Immunophenotype and Analysis of Allo-Reactivity of Umbilical Cord Blood (UCB)-Derived T-Cells Following Ex-Vivo Expansion.

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. The major cause of mortality post transplant is opportunistic infection, due in part to the immunologic naiveté of the UCB T-cells. We have been able to expanded UCB derived T-cells utilizing a skin stromal layer with a supplemental cytokine cocktail. To assist in determining a target population for ex-vivo immunization and to predict the likelihood of inducing GVHD by reinfusion of alloreactive T cells, we identified the immunophenotype of these cells using flow cytometry and assessed their allo-reactivity utilizing mixed lymphocyte culture. The long term goal is to provide UCB-derived adoptive immunotherapy for 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 5,000cGy. 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) (10ng/ml), flt-3 ligand (10ng/ml) stem cell factor (50ng/ml) for 14 days. Interleukin-2 (IL-2) (100u/ml) was then added and the UCB cells remained in culture for a total of 28 days. Cell count, viability, and extensive flow cytometric analysis were performed. In addition we utilized mixed lymphocyte culture to compare unmanipulated and expanded UCB reactivity to patient derived lyphoctyes after the first and second phase of expansion.

Results: Mean fold increases of 9 in CD4+, 11 in CD8+ and 53 in CD4+/CD8+ cells were seen. Of the expanded T cells cells, 81% were CD4+, 20% CD8+ and 3% CD4+/8+. Virtually 100% were alpha-beta T cells. One percent were 15/56+, 79% CD62L(L-selectin)+ and 2.4% CD25(IL-2R)+ . In mixed lymphocyte culture with patient derived lyphocytes, preliminary data suggests that there is no difference in reactivity of the UCB after either the first or second phase of ex-vivo expansion when compared with an unmanipulated UCB fraction.

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, SCF and IL-2 over a patient derived, irradiated skin stromal layer, with maximal expansion in the immature CD4+/CD8+ T-cell subset. The expanded T cells were alpha-beta T cells, which would allow immunization of these cells in standard fashion via co-culture with professional APCs loaded with target antigens. The absence of delta-gamma T cells as well as the small percentage expressing CD 25 is not suggestive of GVHD inducing populations. In mixed lymphocyte culture with patient derived lymphocytes, it does not appear that the expanded cord blood is more alloreactive then unmanipulated cord blood, despite the exposure in culture to patient derived skin fibroblasts and stimulation with cytokines. We hypothesize that the expanded T cells could serve as a target population for adoptive immunotherapy without inducing additional GVHD. Further testing will determine the optimal cellular target and conditions for ex vivo immunization against various viral and fungal antigens. The ex-vivo expanded and immunized T cells could then be reinfused into the patient to augment immune reconstitution and decrease infection related morbidity and mortality in the months following UCBT.