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 6

Reduced A20 function leads to rapid islet allograft rejection.

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Reduced A20 function leads to rapid islet allograft rejection. (A and B)...
(A and B) βTC3 cells cotransfected with an NF-κB.luciferase reporter (A) or an AP-1 luciferase reporter (B) and a CMV.βgal expression construct with or without PCDNA3.1-encoding murine reference A20 or A20 with an I325N coding variant. Cells were stimulated with 200 U/mL TNF for 8 hours (A); for 5, 8, 16, and 24 hours (B); or left untreated. Data represent fold change of stimulated versus nonstimulated and 3 independent experiments. (C) WT BALB/c islets were isolated and transduced with rAd.GFP (+G), rAd.TNFAIP3 (+A) or rAd.TNFAIP3I325N (+I); incubated overnight; and stimulated with 200 U/mL TNF for 0 or 4 hours; gene expression of proinflammatory factors was assessed. Each point represents a well with 300 islets. Data cumulative over 3 independent experiments. (D) Immunoblot (IB) of A20 immunoprecipitated (IP) lysates and whole-cell lysates from βTC3 cells transfected with reference A20 or A20 I325N and IKKγ and lysed following an overnight incubation. Membranes were probed for RIPK1, IKKγ, A20, or β-actin (loading control). (E) Densitometry of coimmunoprecipitated RIPK1 compared A20 pull-down, normalized to reference A20 (WT). Data in D and E represent 2 independent experiments and 3 biological replicates. (F) Expression of TNF-induced genes in islets from Tnfaip3+/+ or Tnfaip3I325N/I325N mice stimulated with TNF for 4 hours. Data shown in cumulative with each point in a column representing an independent islet preparation. (G) Percent of diabetic recipient CBA (H2k) mice normoglycemic following transplantation of 300 homozygous I325N islets (Tnfaip3I325N/I325N (n = 8; mean survival time [MST] = 8), heterozygous I325N (Tnfaip3I325N/+) (n = 8; MST = 13), or WT (Tnfaip3+/+) (n = 8; MST = 13) islet (H2b). (H and I) Islet grafts were excised at postoperative day (POD) 10, and gene expression of known islet derived inflammatory factors (H) and nonislet derived factors (I) measured. Nontransplanted overnight-cultured isolated islets were used as baseline. Statistical significance determined by 1-way ANOVA with Tukey’s multiple comparisons post hoc test (B, C, and F), 2-tailed Student’s t test (E, H, and I), or log-rank test (G). Error bars represent ± SEM or ± SD (E), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001.