Anergic Cells Induced In Vitro By The Blockade Of CD40-CD154 And CD28-B7 Costimulatory Pathways Act As Potent Immunoregulatory Cells

Objective This study is designed to investigate the possibility of inducing mouse anergic cells by blocking the CD40-CD154 and CD28-B7 costimulatory pathways, evaluate whether anergic cells can act as potent immunoregulatory cells in vitro, and observe the cardiac allograft survival after anergic cells injection. The mechanism by which anergic cells regulate immune responses is explored.Methods l.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 for 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. Anergic cells were phenotypically analyzed by the flow cytometry. 2. Heterotopic abdominal cardiac allografting was done in mice and an ideal combination of donor and recipient was chosen among BLAB/c(H-2d), C3H(H-2k) and C57BL/6J(H-2b) inbred strain. 3. In order to accommodate the adoptively transferred cells, all recipient mice were irradiated at a dose of 3.0 Gy before heterotopic abdominal cardiac transplantation. Anergic cells were intravenously injected into 3.0-Gy y-irradiated BALB/c mice immediately after transplantation. To prolong allograft survival, recipient mice injected with anergic cells received Rapamycin therapy (Img/day/kg) for 14 days. Graft survival in different experimental groups was compared using the log-rank test. On day 7 after cardiac transplantation, the subsets of peripheral blood T lymphocytes were analyzed by double labeling procedure. The rejection of cardiac allografts was graded by transplant immunologists and the phenotype of infiltrated lymphocytes in the allografts was also compared with pathologic parameters. Furthermore, the mRNA expression of Thl and Th2 cytokines in the allografts was detected by semi-quantity RT-PCR.Results 1. Primary MLR cells cultured in the presence of anti-CD 154 and anti-CD80 mAbs were hyporesponsive to original stimulator cells (C3H). The failure to proliferate was accompanied by a seriously impaired IL-2 production which was detected in the culture supernatants. The presence of mAbs in the primary MLR mightexplain the hyporesponsiveness. To exclude this possibility, culture cells were allowed to recuperate in fresh medium without mAbs for 2 days, and restimulated with C3H splenocytes for 4 days. Furthermore, hyporesponsiveness was not due to deletion because antigenic restimulation in the presence of exogenous IL-2 led to a response of anergic cells. Thus, combined mAbs blocking of the costimulatory ligands in the primary polyclonal MLR induced genuine anergic cells. Anergic cells strongly suppressed the proliferation of naive BALB/c splenocytes against the original (C3H) stimulator in a dose-dependent manner, but they failed to suppress the proliferation of naive BALB/c splenocytes against the third-party (C57BL/6J) stimulator. The anergic state was reversed by both original (C3H) stimulator and the addition of exogenous IL-2 , while only a slight response was observed with either antigen or IL-2 alone. An increased number of CD25+CD4+T cells was observed in anergic cells compared with control cells cultured in the absence of mAbs. 2.An abdominal heterotopic cardiac transplantation model was established successfully and the survival time was beyond 100 days between donor and reciepient from the same strain mice. The combination of BLAB/c mice as recipients and C3H as donors was chosen for in vivo studies. 3. In in vivo studies, untreated irradiated BALB/c mice rejected C3H cardiac allografts with a median survival time (MST) of 9 days. Those mice injected with the anergic cells and with Rapamycin therapy alone rejected the allografts with MST of 11 and 17 days, respectively. The protocol based on anergic cells injection plus Rapamycin therapy could prolong allograft survival significantly (MST 28 days,...