Novel mechanisms of suppressor activity exhibited by cytotoxic regulatory T cell lines, HOZOT

Objective

Regulatory T (Treg) cells, which play a central role in maintaining immune tolerance, can be grouped into different subtypes, such as naturally occurring Treg, type-1 T regulatory, and Th3. The suppressor activities of Treg cells are mediated through several molecular mechanisms, including immunosuppressive cytokines, cell surface molecules, and cytolytic molecules. In a previous report, we described a novel regulatory human T-cell line (termed HOZOT). The line was established by cocultivating human umbilical cord blood cells with mouse stromal cells in the absence of exogenous cytokines. In this study, we investigated the mechanism of HOZOT's suppressor activity.

Materials and Methods

Suppressor activity of HOZOT was evaluated in vitro by assessing their inhibition of allogeneic mixed lymphocyte reaction, in which CD4⁺CD25⁻ responder T cells were stimulated by dendritic cells (DCs). Responder T cells as well as DCs were prepared from umbilical cord blood using magnetic-activated cell sorting separation system. DNA microarray analysis was performed to search for specific molecules involved in HOZOT's suppressor mechanisms.

Results

We confirmed that suppressing effects were observed in all three subpopulations of CD4/CD8 phenotype. We ruled out possible involvement of HOZOT's cytotoxic activity as well as participation of surface molecules, including cytotoxic T-lymphocyte-associated protein-4, programmed death-1, and glucocorticoid-inducible tumor necrosis factor receptor in suppressor. The supernatant obtained from HOZOT and DC coculture revealed mixed lymphocyte reaction inhibitory activity, indicating the presence of a soluble factor, which mediates suppressor function. Blocking experiments demonstrated that interleukin-10 and transforming growth factor-β were not responsible factors.

Conclusions

HOZOT exerted suppressor activity in the absence of cell contact mechanisms, which are distinct from those of naturally occurring Treg, type-1 T regulatory, and Th3.