CD40-CD154 co-stimulation blockade improves blood glucose control in syngeneic marginal islet transplantation

Y. Koshizuka, K. Yamashita, M. Watanabe, D. Kuraya, M. Ogura, T. Yoshida, H. Kamachi, M. Matsushita, M. Ozaki, S. Todo
Hokkaido university, General surgery, Sapporo, Japan

Objective: Uncontrollable graft destruction elicited by non-specific inflammatory reactions following pancreatic islet transplantation (PITx) has remained unresolved problem. CD40-CD154 costimulation blockade strongly inhibits allo-immune responses, and markedly prolongs allograft survival in rodent and non-human primate PITx models. This costimulatory signaling pathway is also involved in inflammation and innate immune responses. We examined the effect of CD40-CD154 costimulation blockade on early graft loss using a murine syngeneic marginal PITx model.
Methods: Male C57BL/6 mice (B6) served as islet donors and recipients. Handpicked 175 islets (marginal mass volume) were transplanted into the liver of STZ-induced diabetic recipient. Adenovirus vector coding either β-galactosidase (AdLacZ) or CD40Ig (AdCD40Ig) gene was administered to recipient on day -2 (1x10⁸ PFU/body, i.v.). The liver samples were obtained 3 hours after PITx, and tissue expression level of pro-inflammatory cytokines was quantified by real-time RT-PCR (n=6). Surface CD40 expression of islets was analyzed by flow cytometry (n=4).
Results: CD40 expression level of isolated islets immediately after isolation was 11.2±2.7%. When cultured under cytokines (TNFα+IL-1β+IFN-g), CD40 expression level significantly increased from 9.1±2.2% to 28.2±6.8% (p<0.01). Following PITx with 175 islets, none of recipient mice became normoglycemic in both untreated (n=11) and AdLacZ-treated (n=7) control animals. In a sharp contrast, a single infusion of AdCD40Ig significantly cured diabetes in 5 of 6 (83%) recipients (Figure). Liver tissue analyses revealed that AdCD40Ig treatment significantly suppressed TNFα (3.3±1.3 vs. 1.8±0.6, p=0.03), IL-1β (5.6±1.8 vs. 2.9±0.6, p<0.01) and MCP-1 (2.6±1.1 vs. 1.2±0.4, p=0.01) mRNA expression levels as compared to those of AdLacZ treatment.

Conclusion: Pro-inflammatory cytokines promote CD40 expression on islet grafts. CD40-CD154 co-stimulation blockade suppresses inflammatory responses and prevents early graft loss after PITx.