Background This study was to evaluate whether anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro and prolong cardiac allograft survival after adoptive transfer KH*2/5DMethods Anergic cells were induced in vitro by the addition of anti-CD154 and anti-CD80 monoclonal antibodies (mAbs) to primary MLR (mixed lymphocyte reaction) consisting of BALB/c as responder and C3H as stimulator Anergic cells were added to a newly formed MLR in assessing the regulatory capacity and antigen specificity of anergic cells The ability of anergic cells to respond to antigen and/or exogenous recombinant mouse interleukin-2 (rmIL-2) was tested For in vivo studies, anergic cells were intravenously injected into 3 0-Gy γ-irradiated BALB/c mice immediately after heterotopic abdominal cardiac transplantation To prolong allograft survival, recipient mice injected with anergic cells received rapamycin therapy (1 mg·day -1 ·kg -1 ) KH*2/5DResults Anergic cells strongly suppressed the proliferation of naǐve BALB/c splenocytes against the original (C3H) stimulator in a dose-dependent manner, but they failed to suppress the proliferation of naǐve BALB/c splenocytes against the third-party (C57BL/6J) stimulator The anergic state was reversed by both original (C3H) stimulator and additional exogenous IL-2 In in vivo studies, untreated irradiated BALB/c mice rejected C3H cardiac allografts with a mean survival time of (8 6±1 1) days, whereas those injected with the anergic cells rejected the allografts with a mean survival time of (11 8±1 9) days, which was slightly longer than that of the untreated mice The protocol based on anergic cells injection plus rapamycin therapy could prolong allograft survival significantly [(29 6±4 4) days] Conclusions Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro , and prolong cardiac allograft survival after adoptive transfer in the presence of rapamycin therapy This procedure might be clinically useful for prolonging allograft survival if optimal protocols are developed
