Intact active bone transplantation synergizes with anti-CD40 ligand therapy to induce B cell tolerance

Blockade of T cell costimulatory pathways can result in the prolongation of allograft survival through the suppression of Th1 responses; however, late allograft rejection is usually accompanied by an emerging allograft-specific humoral response. We have recently determined that intact active bone (IAB) fragments transplanted under the kidney capsule can synergize with transient anti-CD40 ligand (CD40L) treatment to induce robust donor-specific allograft tolerance and suppress the alloantibody response. In this study, we take advantage of the ability of galactosyltransferase-deficient knockout (GT-Ko) mice to respond to the carbohydrate epitope, galactose-alpha1,3-galactose (Gal), to investigate whether IAB plus transient anti-CD40L therapy directly tolerize B cell responses. GT-Ko mice tolerized to Gal-expressing C3H hearts and IAB plus transient anti-CD40L therapy were challenged with pig kidney membranes that express high levels of Gal. The anti-Gal IgM and IgG responses were significantly suppressed in IAB-tolerant mice compared with controls, while the non-Gal anti-pig Ab responses were comparable. The anti-pig T cell cytokine response (IFN-gamma and IL-4) was comparable in IAB-tolerant and control mice. The tolerant state for the anti-Gal IgM response could be reversed with repeated immunization, whereas the tolerant state for the IgG response was robust and resisted repeated immunization. These observations provide an important proof-of-concept that adjunct therapies can synergize with anti-CD40L Abs to tolerize B cell responses independent of their effects on T cells. This model, which does not require mixed chimerism, provides a unique opportunity for investigating the mechanism of peripheral tolerance in a clinically relevant population of carbohydrate-specific B cells.