Abstract

Background: Viral infections cause significant morbidity & mortality in patients undergoing unrelated allogeneic BMT transplantation before immune reconstitution is completed. It poses a greater risk for recipients of unrelated umbilical cord blood (uUCB) transplants as there is no established antiviral immunity in naïve UCB lymphocytes available for adoptive transfer. UCB T cells also lack Th1/Tc1 cytokines, Granzymes & Perforin which are prerequisites to control viral pathogens. Another major limitation of uUCBT is the lack of donor cells available for post-transplant donor leukocyte infusions (DLI) to boost immunity or induce GVL. However, a fraction of the uCB graft could be available for T cell expansion. In this study we evaluated the feasibility of ex vivo expansion of UCB T cells. We postulated that following expansion naïve T cells may mature & acquire a phenotype compatible with effector function as assessed by expression of essential cytokines & de novo expression of members of the granzyme-perforin pathway.

Methods: Thawed UBC research samples with a leukocyte content <5% of an average UCB graft are processed. T cells are enriched with “EasySep” (StemCell Tech) to deplete CD14, CD16, CD19, CD56, & glycophorin A + cells. 5–7.5*105 T cells/ml are incubated with “CD3/28 T cell Expander” artificial APC beads (Dynal) in X Vivo-15 (BioWhittacker) + 200u/ml IL2 & 10% human serum in gas permeable bags. The initial purity of the T cells is 77–92%. The starting absolute T cell numbers ranged from 0.75 to 2*106 cells. Media & cytokines are added every other day to maintain a concentration of <2*106 cells/ml.

Results: At the end of 14 days UCB T Cells expanded 67 fold +/− 36, n=6. There are significant alterations in phenotype over the 2 weeks (Table 1) with up to 40% of T cells in cell cycle. Compared to the starting resting UCB T cells the majority of expanded cells have acquired the phenotype of activated (HLA-DR+, CD25+ T cells) memory cells, at the expense of naive/recent thymic immigrants (CD45RA+/CD62+). There is an inverted CD4/CD8 ratio due to the higher expansion rate of CD8 T cells (p=0.0035) while there is no difference in apoptosis (p=0.57). However, they all retain expression of CD28 (96% ±8%) along with CD27. Although some T cells have acquired the capacity to secrete granzymes A and B these are still almost a log below normal adult peripheral blood (PB) values & perforin has not been detected. Similarly, while post expansion significantly more T cells secrete cytokines upon PMA + ionomycin stimulation (Table 1) they are below levels of adult PB.

Conclusions: From our preliminary results we can demonstrate effective expansion & partial maturation of UCB T Cells. For example, if one starts with 2*106 total T cells & expands them 67 fold this could provide for DLI ~5*106 T cells/kg for an average pediatric patient (25kg). We are further optimizing & characterizing this model for T cell activity & repertoire. In sum, ex vivo expansion with CD3/CD28 co-stimulation may provide clinically relevant numbers T cells available for adoptive immunotherapy that have also undergone partial maturation.

Characterization of Expanded T Cells as % of all Lymphocytes

VariableMedianSD
CD3+ 99.8 0.1 
CD4+ 35 11 
CD4+/CD8+ 2.3 2.8 
CD45RA+/RO− 13 11 
CD45RO+/RA− 55 22 
CD25+ 42 21 
CD45RA+/CD62+ 38 20 
CD45RA+/CD27+/CD8+ 52 15 
CD45RA−/CD27+/CD8+ 46 15 
KI67/CD8+ 42 
Ki67/CD4+ 32 
HLA DR+ 40 13 
Granzyme A/CD8+ 54 18 
Granzyme B/CD8+ 
Perforin/CD8+ 
VariableMedianSD
CD3+ 99.8 0.1 
CD4+ 35 11 
CD4+/CD8+ 2.3 2.8 
CD45RA+/RO− 13 11 
CD45RO+/RA− 55 22 
CD25+ 42 21 
CD45RA+/CD62+ 38 20 
CD45RA+/CD27+/CD8+ 52 15 
CD45RA−/CD27+/CD8+ 46 15 
KI67/CD8+ 42 
Ki67/CD4+ 32 
HLA DR+ 40 13 
Granzyme A/CD8+ 54 18 
Granzyme B/CD8+ 
Perforin/CD8+ 

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