Novel Double Labeled Mixed Lymphocyte in Vitro Assay to Predict the Dominant Graft in 2 Unit UCB Transplantation

Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/−23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells.

In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO₂. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1.

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We noted that T cells from UCB are primarily at naïve stage as determined by CD45RA (93.8 +/− 7.11%) and CCR7 (84.9 +/− 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 μM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-γ release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-γ by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-γ was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-γ is currently under investigation.

UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow.

No relevant conflicts of interest to declare.