*Purpose: For decades, blockade of CD40-CD154 has been shown to be a highly effective means of inhibiting alloreactive T cell responses and inducing long-term graft survival in both murine and non-human primate models.Unfortunately, thromboembolic complications stymied the clinical translation of CD154 blockers, and instigated the targeting of CD40 as an alternative therapy. However, using a fully allogeneic murine transplant model, we found that CD40 blockade (clone 7-E1G2b) was not as effective in prolonging graft survival as was CD154 blockade (clone MR-1)(p<0.0001), raising the possibility that CD154 binds a second receptor. Moreover, recently reported biochemical evidence suggests that CD11b can function as a second receptor for CD154. Thus, here we interrogatedthe impact of blocking CD154-CD11b interactions during transplantation, both alone and in the context of CD40 blockade.

*Methods: We first queried the impact of CD154 antagonism in the absence of CD40. We also utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154-CD40 interactions.

*Results: Data indicated that anti-CD154 functioned to reduce graft-infiltrating CD8⁺T cells in both WT and CD40^-/- hosts (p<0.05 comparing untreated vs. anti-CD154), thus definitively demonstrating CD40-independent effects of CD154. Recently reported biochemical evidence suggests that CD11b can function as a second receptor for CD154. Thus, we next interrogatedthe impact of blocking CD154-CD11b interactions during alloimmunity. We utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154-CD40 interactions. CD154:CD11b antagonism significantly increased the efficacy of anti-CD40 in prolonging graft survival as compared to anti-CD40 alone (MSTs undefined vs. 21d, respectively, n=9/group, p<0.01). Mechanistically, CD154:CD11b antagonism in the context of CD40 blockade functioned to reduce the number of graft-infiltrating CD8⁺T cells (1.9×10³vs. 4.1×10³, respectively, p<0.05) and CD11b⁺F4/80⁺myeloid cells (1.0×10⁴vs. 3.2×10⁴, respectively, p<0.05) relative to CD40 blockade alone.

*Conclusions: Taken together, these data therefore demonstrate that blocking the ability of CD154 to interact with both CD40 and CD11b is required for optimal inhibition of alloimmunity. As anti-CD40 mAbs move through the pipeline for clinical translation, understanding the impact of CD154:CD11b interactions (that proceed unimpeded in the setting of CD40 blockade) is critical in order to devise innovative strategies to block these interactions and optimize the use of CD40 blockers in the clinic.

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