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 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 30-Gy γ-irradiated BALB / c mice immediately after heterotopic abdominal cardiac transplantation To prolong allograft survival, recipient mice injected with anergic cells received rapamycin ther apy (1 mg · day -1 · kg -1) KH * 2/5 Results 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 studied in untreated irradiated BALB / c mice rejected C3H cardiac allografts with a mean survival time of (8 6 ± 1 1) days, among 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 a blocker of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immu [(29 6 ± 4 4) days] Conclusions Anergic cells induced by the blockade of CD40- noregulatory 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