Background: Islet xenotransplantation is a promising alternative to allo-islet transplantation to treat Type1 Diabetes. Despite significant advances in transgenic pigs, we still require heavy immunosuppression to prevent rejection. To optimise immunosuppressive protocols, we require a means to more regularly monitor the efficiency of immunosuppression on recipients and prevent graft rejection. Here we present our analysis of the development of a more predictive and more regularly performable method.
Methods: Porcine neonatal islet cell clusters (NICC) from GTKO-hCD55-hCD59 transgenic pigs were transplanted into STZ induced diabetic baboons (n=9). All received anti-CD2 antibody (Abs), tacrolimus, anti-CD154 Ab and Belatacept immunosuppression. Blood samples were collected from recipients before and after transplantation from which counts of blood leukocyte subtypes were monitored using multiplex, multi-panel flow cytometry (Flow). NICC function was assessed by multiple daily blood sugar levels (BSL) and monthly glucose tolerance tests. Additionally, NICC xenograft maturation and function were assessed histologically from biopsies with immune-profiling of NICC graft sites analysed by multiplex immunofluorescence staining of the 4 months-post transplant biopsy and endpoint tissue collection.
Results: All diabetic baboons (n=9) were transplanted with GTKO-hCD55-hCD59 transgenic pig NICC’s and were followed to end point following withdrawal of all exogenous immunosuppression. All grafts demonstrated function and provided normoglycaemia in all animals. Systemic porcine C-peptide was detected from day 30 onwards.  Day 120 liver biopsies showed well preserved morphology with no cellular infiltrate and positive staining for insulin, somatostatin and glucagon (n=7).  Flow using IVD BD true count produced equivalent cell counts compared to standard full blood count (FBC) test demonstrated by Deming regression analysis (95% CI slope = 0.91-1.031, p<0.001). Noticeably, cell count by Flow were completed faster (50 minutes ± 15 versus 150 mins ± 30 for FBC test) and used significantly smaller sample volume (standard 50 µl versus 500 ±50 µl blood for FBC test). Using Abs targeting specific markers for different lymphocytes subtypes, the Flow method provided more sensitivity and specificity in real-time monitoring blood immune profiles in xeno-islets recipients. Flow data showed specific T cell-binding and suppression of anti-CD2 Abs with reduction of unoccupied surface CD2 on T cells in recipient’s blood after anti-CD2 Abs treatment (mean of 73.21 %, 55.26% -91.16%, p <0.001). Long-term NICC survival (n=7) showed significant T cell suppression during the first month post-transplant (36.47%, 13.35-50.75 %) compared to short-term NICC survival (n=2) (176.28%, 171.26 – 181.31 %). Early elevated blood lymphocyte levels (40 -142 %) were also detected in the two recipients that lost NICC function shortly after the withdrawal of immunosuppression. Qualitative analysis of endpoint NICC grafts by multiplex immunofluorescence demonstrated NICC destruction through cell-mediated killing with increased lymphocyte infiltration and cytotoxic CD8+T cell activation.
Conclusion: This study demonstrates the utility and reliability of Flow to be a more helpful tool to study immune status of xeno-islet recipients. It is significantly faster to perform than standard FBC, utilising a much smaller sample allowing for more regular and easily obtainable samples from bottom pricks rather than formal blood collection.