Objective: To study the synergistic effect of ICOS-Ig combined with cyclosporine （CsA） on mouse heart transplantation and explore its therapeutic potential. Methods: ICOS-Ig fusion protein was generated by fusing the extracellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro

ICOS-Ig or IgG was added to the primary MLR cultures （BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells）. The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR

and irradiated C57BL/6 （donor） or C3H （third party） spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo

CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG

ICOS-Ig or CsA. Seventy two hours after transfer

the spleen cells of the mice were harvested for the detection of CD4+CFSE+ and CD8+CFSE+ by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups: no treatment group

control IgG treated group （250 μg i.p. d2

4

6）

ICOS-Ig treated group （250 μg i.p. d2

4

6）

CsA treated group （10 mg/kg i.p. d0-6）

ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results: In primary MLR

ICOS-Ig inhibited T-cell proliferation

（inhibition ratio 58±8.2% in 50 μg/ml）. In secondary MLR

ICOS-Ig specifically inhibited donor spleen cells

which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay

CD4+CFSE+ and CD8+CFSE+ in ICOS-Ig and CsA group was stronger than those in control group

which showed ICOS-Ig and CsA could inhibit the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model

survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover

ICOS-Ig combined with CsA group had even longer engraftment （>100 d） than ICOS-Ig or CsA used alone. In histological examination

it was found that there were congestions and edemas in no treatment and IgG treated recipients

together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions （MLR） and cytotoxic lymphocyte reactions （CTL）. Conclusion: These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts

whereas monotherapy is less effective in this regard. Thus

ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection.