Background: Intestinal transplantation (ITx) is the only curative option in patients with irreversible intestinal failure showing complications in parenteral nutrition. However, the immunological load of the gut makes the rejection rate to be higher than the rest of solid organ transplants (80% within the first 90 days after surgery), the graft loss rate and need for re-transplantation at 1, 5, and 10 years 29%, 50%, and 59%, respectively, and the 10-year survival of only 43%. Therefore, new strategies aiming to down modulate host vs. graft response are crucial to improve ITx long term results. Regulatory T-cells (Tregs) are key players in the induction/maintenance of peripheral tolerance, nonetheless their relevance in ITx in almost unexplored. We study the Treg migration/expansion kinetic and its association with the allograft outcome in an experimental ITx model.

Methods: We used a rat heterotopic and allogenic ITx model in which Brown Norway small bowel (jejunum and ileum) was engrafted into Lewis animals. Recipients were randomly divided into 4 groups (n=5): no immunosuppressant treatment, Rapamycin (2 mg/kg), Fk506 (0.6 mg/kg) treatment for 14 days, and Fk506 (0.6 mg/kg) treatment for 7 days and 7 days without immunosuppression. Blood and intestinal biopsies were taken before graft reperfusion and 3, 7, and 14 days after surgery. Acute cellular rejection (ACR) was diagnosed by clinical/histological findings, gene expression of pro- and anti-inflammatory mediators quantified by RT-qPCR and T-cell chimerism/Tregs frequency analyzed by flow cytometry.

Results: Rapamycin and non-treated animals showed significant clinical and histological deterioration since day 7 post-transplant (mild-moderate ACR at day 7 and severe ACR at the days 10-12, p<0.05). The allogenic response was associated with loss of T-cell chimerism (p<0.05), diminished graft Treg frequency (p<0.01) and increased mRNA expression of MCP-1, IL-6, TNF, IFNγ, CCL11, CXCL-10, IDO, and TGFβ (p<0.05). On the other hand, Fk506 used as a first line treatment not only controlled the allogeneic response better than Rapamycin (no significant clinical or histological changes were observed at day 14 after surgery), but also enabled a more gradual exchange of donor/recipient lamina propria T-cell compartment and the expansion of both blood and graft Tregs (p<001). Interestingly, animals that received Fk506 during 1 week and then remained 7 days without immunosuppression, a period proven sufficient for developing an allogeneic response and subsequent tissue damage, still preserved increased Tregs frequency and remained free of ACR. Of note, in these group none of the analyzed genes showed increased mRNA expression at day 14 post-transplant.

Conclusion: In our animal model increased Treg frequency was associated with graft protection even when immunosuppression was withdrawn. Thus, strategies promoting their expansion would be desirable to improve long-term outcomes.