Intact Active Bone Transplantation Synergizes with Anti-CD40 Ligand Therapy to Induce B Cell Tolerance

Abstract

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-α1,3-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-γ 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.

Free Bone Graft Attenuates Acute Rejection and in Combination with Cyclosporin A Leads to Indefinite Cardiac Allograft Survival

“…The free bone grafts were prepared by a method modified from the previous description by Yin et al (36). Briefly, the knee joints containing the heads of tibiae and femora from the hind legs of C3H donor mice or third party C56BL/6 mice were harvested, cleaned of connective tissue, and cut into small fragments.…”

“…To apply this concept to a noncomposite allograft system, previous studies have used "intact active bone" fragments to provide HSCs/MSCs population in its naturally residing environment. It was demonstrated that implantation of such bone grafts under the kidney capsule of recipient mice could synergize with the anti-CD154 Ab to prolong donor organ survival (35,36). To enable a higher number of active cells to be implanted, as well as to develop a clinically relevant procedure, we adopted a technique to implant free bone into the s.c. fat of the recipient animal, the procedure referred to as "free bone cotransplantation".…”

Peripheral deletion of mature alloreactive B cells induced by costimulation blockade

“…Blocking CD154-CD40 interaction also prevents the T-dependent humoral responses (18). As a result, alloantibody production is inhibited (19), which can also be important for the long-term survival of allografts. However, the detailed mechanism of how alloreactive B cells are regulated by such costimulation-targeted therapies is not clear.…”

Long-Term Control of Alloreactive B Cell Responses by the Suppression of T Cell Help

“…Early studies indicated that anti-CD154 treatment could suppress B cell responses, but not induce B cell tolerance (36). In contrast, we have reported that anti-CD154 therapy, in the context of allograft tolerance models, can induce the long-term suppression of alloantibody and anti-Galactose-␣(1-3) Galactose carbohydrate moiety Ab production (37). To address the different effects of anti-CD154 on B cell responses, we have examined the basis for how alloreactive B cell responses are controlled in a mouse heart allograft transplant model.…”