HOZOTs, novel human regulatory T-cell lines, exhibit helper or suppressor activities depending on dendritic cell or anti-CD3 stimulation

Objective

HOZOT cell lines (HOZOTs) are a new type of regulatory T cells established from human umbilical cord blood without using cytokines. In addition to their unique FOXP3⁺CD4⁺CD8⁺CD25⁺ phenotype, HOZOTs are bifunctional and can exert either suppressor or cytotoxic activities. To further characterize HOZOTs, we cocultured HOZOTs with responder T cells under different stimulation conditions and found another function of HOZOTs.

Materials and Methods

Naïve CD4⁺T cells as responder cells were stimulated with dendritic cells or plate bound anti-CD3 antibody. As effector cells, HOZOTs were added to this culture and proliferation of the responder cells were monitored by ³H-thymidine incorporation or carboxyfluorescein succinimidyl ester dilution method. To investigate the molecular mechanisms, antibodies specific for interleukin (IL)-2/IL-2R or cell surface molecules were used for blocking experiments.

Results

The proliferation of naïve CD4⁺T cells was suppressed by one HOZOT line, HOZOT-4, when the responder cells were stimulated with dendritic cells. However, responder cell proliferation was augmented by HOZOT-4 when these cells were stimulated with anti-CD3 antibody. This opposing function to responder cells was unique to HOZOTs because naturally occurring regulatory T cells suppressed proliferation of both dendritic cell– and anti–CD3-antibody-stimulated cells. IL-2 was not involved in the mechanism of the helper activity of HOZOT-4 as blocking antibodies for IL-2 and IL-2R did not abrogate the helper activity. Moreover, this helper activity could not be reduced by blocking costimulatory pathways such as CD28/B7, CD4/human leukocyte antigen–DR, and intercellular adhesion molecule–1/lymphocyte function-associated antigen–1.

Conclusion

We demonstrated a new function of HOZOTs as helper T cells in addition to suppressor and cytotoxic activities, characterizing HOZOTs as multifunctional T cells.