IMMUNE REGULATION AND TOLERANCE I

R. Sucher¹, R. Oberhuber¹, N. Fischler², I. Kronberger¹, R. Öllinger¹, S. Schneeberger², D. Fuchs³, E. Werner¹, B. Zelger⁴, G. Tellides⁵, N. Pilat⁶, T. Wekerle⁶, R. Margreiter², J. Pratschke¹, G. Brandacher^1
1Department Of Visceral, Transplant And Thoracic Surgery, Innsbruck Medical University, Innsbruck/AUSTRIA, ²Department Of Visceral Transplant And Thoracic Surgery, Innsbruck Medical University, Innsbruck/AUSTRIA, ³Department Of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck/AUSTRIA, ⁴Department Of Pathology, Innsbruck Medical University, Innsbruck/AUSTRIA, ⁵, Yale University School of Medicine, New Haven/UNITED STATES OF AMERICA, ⁶Department Of Surgery, Medical University Vienna, Vienna/AUSTRIA

Body: Introduction: Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well established tolerance induction strategy. Although previous in vitro studies confirm that CTLA4Ig up-regulates IDO expression in DCs, the precise mechanisms of CTLA4Ig and IDO interaction remain unclear. Here we studied if concerted immunomodulation in vivo by CTLA4Ig, IDO and Tregs accounts for indefinite survival of murine cardiac allografts. Methods: C57BL/6 IDO (WT/knock outs) mice received BALB/c hearts. Group 1 [No treatment], Group 2 [Donor-specific transfusion (DST)], Group 3 [CTLA4Ig], Group 4 [CTLA4Ig+DST], Group 5 [CTLA4Ig+DST+ IDO inhibitor 1-Methyl-Tryptophan (1-MT)] and Group 6 [CTLA4-Ig+DST+ aCD25 mAb]. 1-MT was delivered in slow release pellets (at surgery or POD 50). Serum enzyme activity of IDO (kyn/trp) was analyzed by HPLC. Quantitative PCR was used for mRNA expression of IDO1/IDO2, Foxp3 and granzyme B. Anti-donor Abs were screened by FACS. Histopathology (H&E) and immunohistochemistry (for IDO, Foxp3, CD4, CD8, CD20, CD68 and C4d) of tissues was performed. Results: Graft survival: Group 1 [7.7±1.9 d], Group 2 [10.7±1.3 d], and Group 3 [47.7±29.8 d]. Group 4: Indefinite graft survival [>100 d] and tolerance without chronic rejection in IDO WT but acute rejection in IDO knock out recipients. Group 5: IDO inhibition with 1-MT, either at transplant or at POD 50, abrogated CTLA4Ig+DST tolerance induction. Group 6: aCD25 mAb depletion of Tregs prevented CTLA4Ig+DST tolerance induction. Tolerant recipients had significantly higher IDO activity as compared to non-tolerant animals, which markedly correlated with intragraft IDO and Foxp3 levels on immunostaining. IDO1/IDO2 mRNA expression was similar in tolerant and non-tolerant recipients. Anti-donor Abs were absent in all long-term survivors. Conclusion: This study provides the first direct in vivo evidence that CTLA4Ig induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.

Disclosure: All authors have declared no conflicts of interest.