CD154:CD40 Co-Stimulatory Blockade: A Specific Approach To Prevent Sensitization to Donor Alloantigens.

Introduction: Recipient sensitization is one of the most critical problems facing clinical transplantation. Patients with sickle cell disease are presensitized to HLA-alloantigens from chronic transfusion therapy. These allosensitized recipients experience a higher failure rate in engraftment and require more intense conditioning. A method to prevent sensitization would have a significant impact on transplant outcomes in these recipients. We have identified that B-cell responses and humoral immunity are the dominant factor in sensitization to MHC alloantigens. T-cell-dependent B-cell responses require physical cell:cell contacts that drive the different steps of B-cell differentiation to memory B-cells or to Ab-producing cells. Several signal pathways have been identified that are important for initiation of B-cell differentiation, including costimulation through the CD28/B7 or TNF/TNFR families. Here, we examined whether blocking the costimulatory interaction between T- and B-cells with anti-CD154, anti-OX40L, or anti-ICOS during exposure to alloantigen would prevent allosensitization. Methods: B6 (H-2b) mice received allogeneic BALB/c (H-2d) skin grafts on day 0 and were treated with anti-CD154 (day 0 and day +3), anti-OX40L (day 0 to +4), or anti-ICOS (day +4, 6, 8 and 10). Anti-donor specific antibodies were measured by flow cytometry cross-match assay 4 weeks after skin grafting and reported as mean fluorescence intensity (MFI). Results: Recipients treated with anti-CD154, anti-OX40L, or anti-ICOS rejected BALB/c skin grafts with a time course similar to normal B6 controls (12.7±2.4 days). The anti-donor antibody titer in mice treated with anti-CD154 (5.9±3.9) was slightly, but not significantly ( P >0.05) higher than for naive mice (3.8±0.6). Levels of allospecific Ab were markedly higher in mice treated with mAb anti-ICOS (123.6±38.1; P <0.001) and anti-OX40L (80.6±23.3; P <0.0001) compared with naive controls or anti-CD154 treated animals. The Ab level in mice treated with mAb anti-ICOS was similar to sensitized controls (skin grafting only without mAb treatment; MFI: 132.6±38.5; P =0.69). The anti-donor Ab in mice treated with mAb anti-OX40L was significantly lower ( P =0.02) compared to sensitized controls, but the difference was much more significant when compared with naive controls. To evaluate sensitization in vivo , mice were conditioned with 950 cGy and transplanted with 15×106 of BALB/c BM cells 5–7 weeks after skin grafting. As expected, engraftment did not occur in sensitized control mice (no Ab treatment). Engraftment also did not occur in mice treated with anti-ICOS. Only 16.7% of mice engrafted with anti-OX40L treatment. In contrast, 75.0% of mice engrafted with anti-CD154 treatment. Conclusion: Blocking molecular interactions between CD40/CD154 abrogated the generation of antibody against donor antigens. The CD154 blockade effect on Ab production was specific and not a general feature of co-stimulatory blockade, as blockade of ICOS or OX40L did not completely inhibit Ab generation. These data suggest that the engagement of B-cell molecule CD40 and its ligand CD154 plays a central role in T-cell-dependent B-cell responses. These findings could have a significant impact on management of sensitized recipients in the clinic. ![Figure][1] Figure [1]: pending:yes