Noninvasive diagnosis of recurrent autoimmune type 1 diabetes after islet cell transplantation

Abstract

Islet cell transplantation is curative therapy for patients with complicated autoimmune type 1 diabetes (T1D). We report the diagnostic potential of circulating transplant islet-specific exosomes to noninvasively distinguish pancreatic β cell injury secondary to recurrent autoimmunity vs immunologic rejection. A T1D patient with hypoglycemic unawareness underwent islet transplantation and maintained normoglycemia until posttransplant day 1098 before requiring exogenous insulin. Plasma analysis showed decreased donor islet exosome quantities on day 1001, before hyperglycemia onset. This drop in islet exosome quantity signified islet injury, but did not distinguish injury type. However, analysis of purified transplant islet exosome cargoes showed decrease in insulin-containing exosomes, but not glucagon-containing exosomes, indicating selective destruction of transplanted β cells secondary to recurrent T1D autoimmunity. Furthermore, donor islet exosome cargo analysis showed time-specific increase in islet autoantigen, glutamic acid decarboxylase 65 (GAD65), implicated in T1D autoimmunity. Time-matched analysis of plasma transplant islet exosomes in 3 control subjects undergoing islet cell transplantation failed to show changes in islet exosome quantities or intraexosomal cargo expression of insulin, glucagon, and GAD65. This is the first report of noninvasive diagnosis of recurrent autoimmunity after islet cell transplantation, suggesting that transplant tissue exosome platform may serve as a biomarker in islet transplant diagnostics.

Keywords: B cell biology; autoantigen; basic (laboratory) research/science; insulin/C-peptide; islet transplantation; molecular biology; rejection; translational research/science.

© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

FIGURE 1

Glycemic control and recurrence of diabetes in patient A over posttransplant follow-up is shown. A, Exogenous insulin requirement, hemoglobin A1c, and autoantibody GAD65 titers are shown. After maintaining normoglycemia post-islet cell transplantation, patient A developed GAD65 autoantibody at 33 months (1001 d); and at 36 months (1098 d), fasting hyperglycemia was noted, requiring resumption of insulin therapy. B, β cell secretory capacity derived from the acute insulin response to arginine stimulation under hyperglycemic clamp conditions provided a measure of the functional allogeneic islet mass yearly after transplant. In Patient A, β cell secretory capacity was near-normal and stable at 12 and 24 months, but declined by 58% at 36 months, when the patient developed recurrent diabetes. GAD65, glutamic acid decarboxylase 65

FIGURE 2

Change in circulating transplant islet exosome signal correlates with rise in GAD65 antibody titer. A, Nanoparticle detector analysis for transplant islet-specific exosomes (TISE) signal in Patient A over the follow-up is shown. Blue represents total plasma exosomes, and the red curve represents TISE signal characterized using anti-donor HLA-specific antibody quantum dot on the fluorescence mode. Compared to the pretransplant sample, TISE were seen at 60 minutes post-islet cell transplantation, and detectable over long-term follow-up. Day 1001 sample showed a decrease in TISE signal to below time-matched islet transplant controls with long-term insulin independence (not shown). B, Antibody titer to islet autoantigen, GAD65, in Patient A is shown. The first increase in GAD65 antibody was noted from day 1001 sample, at the same time point when TISE signal drop was noted in Patient A compared to time-matched controls. GAD65, glutamic acid decarboxylase 65

FIGURE 3

Intraexosomal mRNA cargo of donor islet-specific exosomes reflects endocrine cell-specific constituents of the functional transplanted islet mass. Circulating donor islet-specific exosomes were enriched from recipient plasma in Patients A, B, C, and D using anti-donor HLA I-specific antibody conjugated beads. Reverse transcription polymerase chain reaction analysis for insulin and glucagon mRNA cargoes in transplant islet exosomes at follow-up time points is shown for all 4 patients (A-D). In all cases, IgG isotype beads (negative control) from posttransplant samples and pretransplant samples did not show any enrichment of insulin or glucagon in the samples. In Patients B, C, and D, insulin and glucagon mRNAs were detected in all posttransplant samples, with similar ratios of expression between these 2 endocrine markers, suggesting stable β and α cell function, respectively, in the transplanted islets. In Patient A, day 56 sample showed high expression of insulin and glucagon mRNAs, but days 455, 1001, and 1197 samples showed progressive loss of insulin expression without change in glucagon expression, demonstrating selective loss of β cell exosome output secondary to selective β cell destruction in the transplanted islets with preserved transplant islet α cell function. β-actin mRNA control is also shown

FIGURE 4

Recurrent diabetes in Patient A is associated with upregulation of islet self-antigen GAD65 in circulating transplant islet exosomes. Because exosomes are implicated in immune regulation, donor islet-specific exosome cargo was analyzed for expression of T1D self-antigen, GAD65, using anti-donor HLA I-specific antibody conjugated beads. A, Reverse transcription polymerase chain reaction for GAD65 mRNA expression showed its absence in controls, Patients B and C, but selective upregulation was seen in days 455 and 1197 samples in Patient A. This patient developed GAD65 autoantibody on day 1001, suggesting that exosome-based expression of selfantigens may precede development of T1D autoantibody. B, Western blot analysis of donor islet-specific intraexosomal cargo also showed selective upregulation of GAD65 protein in Patient A at day 455 time point, but was undetectable in Patient B. Patient C showed trace to undetectable levels of GAD65 protein. GAD65, glutamic acid decarboxylase 65; T1D, type 1 diabetes