Purpose:

We have shown that co-culture of recipient and irradiated donor splenocytes under presence of anti-CD80 and anti-CD86 mAbs generates immunosuppressive cells in-culture and induces tolerance in subhuman primates. In this study, we determined the applicability of the procedure with human cells.

Methods:

Human PBMCs were co-cultured with irradiated allogeneic PBMCs for 2 weeks in the presence of anti-CD80 and anti-CD86 mAbs. The generated cells were examined for their phenotypes and immunosuppressive effect by flowcytometory and MLR to identify the cells in charge for immunosuppression, cells were isolated/depleted from the generated cells according to cell-phenotypes, and inhibitory effect was assessed by MLR.

Results:

Addition of generated lymphocytes inhibited MLR in proportional to added cell number. The MLR inhibition was relatively stronger to the donor- than 3rd party-Ags. By culturing the cells, mainly CD4⁺ T cells increased from 40.3% to 55.0%. In particular, CD25⁺Foxp3⁺ and CD127^loFoxp3⁺ regulatory phenotypes significantly increased from 6.5% and 8.7% to 23.8% and 22.6%, respectively. When CD3⁺ cells were removed from the generated cells, inhibition of MLR was completely lost. Conversely, the effect was restored when added. Both CD4⁺ and CD8⁺ T cells contributed to the suppressive effect. (figure1) Removal of B cells, monocytes, NK cells, dendritic cells (DCs) did not alter MLR inhibition. Further studies in order to identify the cell phenotype are currently undergoing.

Conclusion:

In human, immunosuppressive cells are generated by co-culture of recipient and irradiated donor PBMCs under CD80/CD86 costimulation blockade. CD3⁺ T cells, including both CD4⁺ and CD8⁺ phenotypes are responsible for immunosuppressive effect.

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