The efficacy of adoptive T cell therapy largely depends on the in vivo persistence of infused cells (Robbins PF et al. J Immunol 2004;173 :7125), that is related to the differentiation stage. The less differentiated central memory (CM) T cells display superior proliferation, persistence and anti-tumor response following infusion when compared to the more differentiated effector memory (EM) T cells. However both T cell receptor (TCR) gene transfer and in vitro expansion of antigen-specific T lymphocytes necessitate lymphocyte activation; with lymphocytes from adult donors this results in accumulation of highly differentiated EM CD45RA+ (EMRA) cells with reduced half life and proliferative capability. Cord blood (CB) lymphocytes are predominantly naïve (N), and we have studied the potential utility of these cells for TCR transduction-based immunotherapy.
T cells from CB samples were activated by anti-CD3 antibody plus IL-2, and transduced with a TCR specific for a peptide from the EBV LMP2 molecule using a retroviral vector. Lymphocytes from adult donors were also transduced as controls. We found that after transduction CB and adult lymphocytes showed comparable levels of TCR expression. Moreover, both CB and adult T cells rapidly shifted toward a more mature phenotype, CB cells becoming mainly CM (60%), and adult T cells becoming mainly EM (52%). In keeping with their phenotype, the expression CD57, a marker of replicative senescence was low in CB T lymphocytes (18%) and high in adult T lymphocytes (68%).
To asses if the transduced cells were able to respond to specific antigen stimulation, transduced lymphocytes were incubated with peptide-pulsed autologous dendritic cells. CB T cells expanded as effectively as lymphocytes from adult donors with a 50-fold increase in cell count after 3 week culture, with no indication that the expansion was leveling off. At that time point more than 50% of the cells expressed the transduced TCR both in CB and in adult samples.
Further, CB cultures were dominated by EM accounting for a mean of 55% of the cells at the end of the culture period with very few EMRA. In contrast, adult blood cell cultures were characterised by a marked increase in EMRA which came to comprise a mean of 66% of the final population. Further phenotypic analysis at the end of the in vitro expansion demonstrated that CD27, a marker of less differentiated T cells, was expressed on 85% of transduced CB T cells but only on 39% of adult T cells, confirming that CB lymphocytes had retained a less differentiated phenotype.
To investigate the effector function of the transduced cells we demonstrated that after further stimulations. polyfunctional CB CD8 cells secreting IFNg, IL2 and TNFa increased from 0.8% after the first stimulation to 4.5% after re-stimulation. In contrast, polyfunctional transduced CD8 T cells from adults decreased from 2.0% to 0.9% during the same period. Both transduced CB and adult CD8 cells demonstrated effective HLA-restricted, antigen-specific target cell lysis in a standard chromium release assay.
Our results suggest that TCR-transduced CB T cells have greater in vivo expansion potential, making these cells potentially more suitable for adoptive immunotherapy, particularly in those cases where autologous lymphopheresis or donor lymphocyte infusion is not an option.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.