A20 as an immune tolerance factor can determine islet transplant outcomes
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
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Research Article Inflammation Transplantation

A20 as an immune tolerance factor can determine islet transplant outcomes

Abstract

Islet transplantation can restore lost glycemic control in type 1 diabetes subjects but is restricted in its clinical application by a limiting supply of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signaling thresholds. Here, we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for ~45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon Tregs and was antigen specific, and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signaling, graft hyperinflammation, and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators, with no impact on glucose-stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signaling thresholds to favor immune tolerance and promotes islet allogeneic survival. Clinically, this would allow for reduced immunosuppression and support the use of alternate islet sources.

Authors

Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey

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Figure 2

Improved survival characteristics of an A20-expressing islet allograft.

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Improved survival characteristics of an A20-expressing islet allograft. ...
Primary islet preparations transduced with adenoviral constructs encoding for GFP or human A20 or left noninfected (NI) were (A) lysed in duplicate (1 and 2), with A20 protein levels assessed by immunoblot, or (B) treated with 200 U/mL of TNF for 4 hours and expression of inflammatory factors measured (* represents A20 versus GFP; ^ represents A20 versus NI). Data represent 3 independent islet preparations. (C and D) 300 NI islets (n = 11) or those expressing GFP (n = 9; P = 0.16) or A20 (n = 27; P = 0.002) were transplanted under the kidney capsule of allogeneic C57BL/6 mice and (C) blood glucose levels (BGL) and (D) percent of mice remaining normoglycemic monitored for the indicated days. Significance determined by Log-rank test. (E) Nephrectomies (N) were conducted at postoperative day (POD) 100 for a portion of A20-expressing long-term–surviving islet grafts. (F) H&E staining or insulin labeling (INS) of long-term–surviving (>100 days)grafts, representative of 7 long-term–surviving grafts. (G) Insulin staining of GFP- or A20-expressing grafts at POD 10. Scale bar: 200 μm (4× magnification) and 100 μm for panel inserts (10× magnification), representative of 4 islet grafts per treatment. (H) RNA levels of inflammatory factors from GFP- (closed square) or A20- (closed circle) expressing grafts harvested at POD 10, as well as A20-transduced long-term–surviving grafts harvested at > POD 100 (gray-filled circle). Each point in a column represents an individual islet graft. Nontransplanted overnight-cultured isolated islets were used as baseline. Error bars ± SEM and statistical significance determined by 1-way ANOVA with Tukey’s multiple comparisons post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ^P < 0.05; ^^P < 0.01; ^^^P < 0.001.