Engineering Stromal Cells for Targeted Differentation of Adult Bone Marrow Stem Cells Into Antigen-Specific Helper T Cells

There is growing evidence that CD4 helper T cells play an important role in orchestrating cytotoxic T lymphocytes (CTLs) or CD8 T cells to elicit efficient immune responses by providing cognate help. This “cognate help” appears to be also necessary for achieving a robust and long-lived CTL response against cancer cells. Thus, adoptive transfer of antigen-specific CD4 T cells, along with CD8 T cells, could afford a better approach for generating productive antigen-specific immunity for treatment of infectious diseases and cancer. We developed a murine stromal cell line capable of directing the differentiation of adult bone marrow stem cells transduced to express CD4 T cell receptors into antigen-specific T cells in vitro. Our method relies on a recently reported OP9-DL1 coculture system, in which the ectopic expression of a notch ligand in a bone marrow stromal cell line, OP9, was found to be sufficient to support the development of hematopoietic progenitors into T cells in vitro. However, OP9-DL1 cells lack the expression of MHC class II molecules, which may limit their capacity to support the development of CD4 T cells. This prompted us to also evaluate the effect of the expression of MHC class II proteins in OP9-DL1 cells on generating antigen-specific CD4 T cells. We verified that the yield of T cells generated from bulk adult bone marrow cells cocultured on OP9-DL1 cells was extremely low; most of the development of the cells was halted at the DN2 and DN3 stages. When adult progenitor cells were transduced to express pre-arranged OT2 TCR alpha and beta chains, a majority of the cells (>70%) were committed to the T cell linage within two weeks of cocultures. CD3 and TCR expressions were detected as early as day 9 of cocultures. Several interesting features were observed in T cell development in the TCR transduction cocultures with the expression of the murine MHC class II protein, I-Ab. We first observed different expansion kinetics in the presence of I-Ab. Transduced bone marrow cells started marked expansion earlier (day 9) in OP9-DL1-IAb coculture, but quickly reached a plateau at day 14. In contrast, a slower expansion occurred in the OP9-DL1 coculture at day 10, followed by a rapid expansion after day 17. We therefore observed a reduced expansion capacity of OP9 cells upon an enforced expression of I-Ab. It was also found that I-Ab expression in the OP9-DL1 cell line could accelerate T cell development. Approximately 10% of bone marrow cells became CD3+OT2+ T cells by day 9 of the OP9-DL1-IAb coculture, compared to around 6% of these T cells appearing in the OP9-DL1 coculture. A close examination of the development of DN cells revealed that 52% of OP9-DL1-IAb-cocultured bone marrow cells reached the DN4 stage by day 14, whereas only 21% of the OP9-DL1-cocultured cells were able to arrive at the same stage. Similar acceleration was also observed in the development of single-positive cells. A majority of the bone marrow cells (>45%) turned into CD4+CD8+ SP cells in the OP9-DL1-IAb coculture at day 22, while only 18% of the cells from the OP9-DL1 coculture displayed the SP phenotype. We tested the functional properties of SP CD4 T cells generated from the TCR transduction cocultures. Upon polyclonal anti-CD3/CD28 stimulation, these cells proliferated, displayed typical surface markers of activated T cells, and secreted the cytokines IFN-γ and IL-2, indicating that these T cells reached functional maturity and were able to initiate TCR signaling in response to stimulation. As compared to CD4 T cells developed from the OP9-DL1 coculture, a much higher production of cytokines (IFN-γ and IL-2) were detected for T cells generated from the OP9-DL1-IAb coculture, suggesting that the provision of interaction between the OT2 TCR and I-Ab in the coculture might enhance the functional development of CD4 T cells. In conclusion, we demonstrated an example of in vitro generation of antigen-specific CD4 T cells by retroviral transduction of adult bone marrow cells to express a CD4 TCR. Ectopic expression of a MHC class II protein could accelerate the development of TCR-transduced bone marrow cells, resulting in functionally improved antigen-specific CD4 T cells.