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Improved allogeneic islet graft survival in mice by oxidized Adenosine Tri-Phosphate treatment

Michele Podetta^1,2, Carmen Fotino¹, R. Damaris Molano¹, Judith Molina¹, Elsie Zahr-Akrawi¹, Maite Lopez-Cabezas¹, Susana Villate¹, Fabio Grassi³, Luca Inverardi^1,4,5, Paolo Fiorina⁶, Camillo Ricordi^1,4,5,7,8, Antonello Pileggi^1,5,7,8

¹Diabetes Research Institute, University of Miami, Miami, FL, United States; ²Surgery, University of Pavia - IRCCS San Matteo Hospital, Pavia, PV, Italy; ³The Institute for Research in Biomedicine, Bellinzona, Switzerland; ⁴Medicine, University of Miami Miller School of Medicine, Miami, FL; ⁵Microbiology & Immunology, University of Miami Miller School of Medicine, Miami, FL; ⁶Children’s Hospital, Harvard Medical School, Boston, MA; ⁷Surgery, University of Miami Miller School of Medicine, Miami, FL; ⁸Biomedical Engineering, University of Miami, Miami, FL, United States

Lymphocyte Function-associated Antigen 1 Anti-(LFA-1) plays a key role in lymphocyte trafficking and co-stimulation. Short-course anti-LFA-1 blockade is partially successful in preventing murine allograft rejection in mice. Modulation of early post-transplant inflammation has been recognized to improve engraftment and survival of allografts. Oxidized Adenosine Tri-Phosphate (oATP) is largely utilized as a nonselective blocker of the purinergic (P2X7) receptor with recognized anti inflammatory and analgesic proprieties. Our study aimed at evaluating the effects on islet allograft survival of oATP therapy alone or combined with LFA-1 blockade.

DBA/2 (H2d) islets were transplanted under the kidney capsule of chemically-induced diabetic C57BL/6 mice (H2b). Single or combined treatments included: oATP (Medestea Research) 0.25mg/mouse intravenously daily on day 0-4, then biweekly for 4 weeks; 7-day course of anti LFA-1 antibody (KBA clone) 100ug/mouse/day intraperitoneally from 0.

Naïve splenocytes showed a dose-dependent suppression of proliferative response to mitogenic stimulation (both mixed lymphocyte reaction and anti-CD3) in the presence of increasing concentrations of oATP in vitro. Control animals rejected allogeneic islets with a median of 15 days (n=12). Long-term allograft survival (>100 days) was observed in 25% of animals receiving oATP treatment alone (median survival=63 days; n=8; p=0.04), and in 57% of animals treated with anti-LFA-1 (n=7). Combination therapy based on oATP and LFA-1 blockade yielded 100% long-term graft survival (n=6; p=0.0007 vs. control group).

Our preliminary data shows a direct immunomodulatory effect of oATP both in vitro and in vivo. Furthermore, administration of oATP enhanced the efficiency of LFA-1 blockade in inducing long-term islet allograft survival in a fully MHC-mismatch transplant model. Combinatorial strategies targeting purinergic receptors and co-stimulatory blockade may allow for the development of efficient tolerogenic clinical protocols in the near future.