Introduction: Costimulatory signal pathways, CD28/B7 (CD80/CD86) and CD40/CD154, are important in the immune response to both allo- and xeno-transplantation. However, in vivo observations indicate that CD28/B7 pathway blockade is not as efficient as CD40/CD154 pathway blockade in preventing de novo anti-pig antibody formation in xenograft recipients. The CD40/CD154 pathway plays an important role in T cell-dependent and T cell-independent B cell activation, resulting in increased humoral immunity (i.e., the development of anti-donor antibodies). Here, we review the current understanding of the role of the CD40//CD154 pathway in xenotransplantation and summarize recent mechanistic and preclinical advances in evaluating candidate therapeutic approaches to target this receptor-ligand in xenotransplantation.

Methods: (1) The suppressive capacity on human PBMC proliferation (when stimulated by wild-type pig aortic endothelial cells) by (i) an anti-CD40mAb (2C10) or (ii) human CTLA4-Ig (belatacept) was investigated in vitro. (2) The survival of pig kidney xenografts in NHPs was compared between (i) conventional therapy (based on tacrolimus and CTLA4-Ig) and (ii) an anti-CD40mAb-based regimen. (3) Published reports of kidney allo- and xeno-graft survival between (i) anti-CD40mAb and (ii) anti-CD154mAb regimens were reviewed. (4) Molecular mechanisms of CD40-CD154 signaling in pig cells were assessed by incubation with soluble human CD154 (as a T cell-independent pathway) in vitro.

Results: (1) In vitro blockade of the CD28/B7 costimulation pathway showed more efficacy than blockade of the CD40/CD154 pathway in inhibiting the human anti-pig T cell response (p<0.05). (2) However, in vivo, conventional therapy (including CD28/B7 pathway blockade) was associated with significantly shorter xenograft survival compared to an anti-CD40mAb regimen (p<0.01). Graft failure resulted from acute humoral xenograft rejection (AHXR). (3) There was an increase in survival for both allo- (p<0.0001) and xeno-grafts in NHPs treated with an anti-CD154mAb compared to anti-CD40mAb. (4) Human soluble CD154 directly activated pig cells through NFκB and p38 signaling, resulting in increased inflammatory gene expression, e.g., of IL-8 and MCP-1.

Conclusions: All of the data suggest that therapeutic antibodies directed at CD154 (e.g., anti-CD154mAb) demonstrate most promise for the prevention/treatment of AHXR. In addition, anti-CD154mAb could be beneficial in suppressing T cell-dependent and -independent B cell activation and also pig cell activation.