Abstract 1491

Poster Board I-514


A complete understanding of lymphocyte development, particularly factors driving T and natural killer (NK) cell differentiation from progenitor cells, remains an elusive goal in medicine. T and NK cells are key regulators in the defense against infections and malignancies and play a direct causative role in autoimmune diseases and graft-versus-host disease. The OP9-DL1 stromal line is an important tool in the in vitro study of lymphocyte development. Lymphocyte progenitors (KLS,Thy1.1-) harvested from adult murine bone marrow and seeded on this stromal line can be followed through stages of maturation by immunophenotyping. We observed that addition of stem cell factor (SCF), contaminated with lipopolysaccharide (LPS) through its production in E. coli, was particularly effective at promoting NK cell development in the OP9-DL1 culture system. Toll-like receptors, an important component of anti-microbial defense by the innate immune response, recognize LPS and other microbial products. Toll-like receptor ligands (TLR-L) have been shown to enhance NK cell proliferation, however an effect on NK cell differentiation from progenitor cells has not been established. A separate set of experiments led us to hypothesize that ascorbic acid (vitamin C) promotes T cell differentiation. We therefore designed experiments to evaluate the differential effects of TLR-L and ascorbic acid on NK and T cell development from lymphoid progenitors co-cultured with OP9-DL1 stromal cells.


Lymphocyte progenitor cells (KLS,Thy1.1-) were sorted from adult mouse bone marrow and 1000-2000 progenitor cells were seeded per well in a 24 well plate coated with OP9-DL1 stroma. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/ml), and SCF (100 ng/ml) plus one of 5 different TLR-L (TLR1/2, TLR3, TLR4, TLR5, and a crude LPS preparation that likely contains a number of TLR-L), with or without addition of a stabilized form of ascorbic acid. Cells were passaged, counted and re-seeded with fresh media and supplements twice a week over a 30-day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, T cell receptor beta chain and T cell receptor gamma-delta chains. NK cells were evaluated for the presence of NKp46, NK1.1, and DX5.


We observed robust cell expansion, inhibited somewhat by addition of ascorbic acid. The inhibitory effect of ascorbate on expansion was most pronounced in the culture condition lacking TLR-L. T cell differentiation was markedly advanced by the addition of ascorbic acid in the absence of TLR-L, with the majority of cells co-expressing CD4/CD8 and TCRB/CD3. The addition of different TLR-Ls inhibited T cell differentiation, and this inhibition was partially rescued by addition of ascorbic acid. NK cell differentiation, defined as co-expression of NKp46 and NK1.1, was two to three-fold greater with the addition of TLR1/2, TLR4, TLR5, and crude LPS compared to cultures lacking TLR-L addition. In each of these conditions, NK cell differentiation was markedly inhibited by addition of ascorbic acid.


Our data supports the hypothesis that both T and NK cell progenitors require Notch signaling for differentiation. In our in vitro model, differentiation of one lineage at the expense of the other can be manipulated with addition of TLR-L or ascorbic acid. Addition of bacterial TLR-L promotes NK cell differentiation at the expense of T cell differentiation; an effect that is partially overcome with the addition of ascorbic acid. The addition of ascorbic acid promotes robust T cell differentiation, and inhibits significant NK cell differentiation in all conditions. The ability of ascorbic acid to promote T cell differentiation appears to dominate over TLR-L promotion of NK lineage differentiation. Further work will include microarray to evaluate these effects at a genetic level. These findings will contribute to our understanding of the immune response under normal and pathologic conditions, and further a model both for study and ex vivo expansion of immune cells for therapeutic use.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.