Type 1 diabetes (T1D) is an autoimmune disease in which pathogenic lymphocytes target autoantigens expressed in pancreatic islets, leading to the destruction of insulin-producing β-cells. Zinc transporter 8 (ZnT8) is a major autoantigen abundantly present on the β-cell surface. This unique molecular target offers the potential to shield β-cells against autoimmune attacks in T1D. Our previous work showed that a monoclonal antibody (mAb43) against cell-surface ZnT8 could home in on pancreatic islets and prevent autoantibodies from recognizing β-cells. This study demonstrates that mAb43 binds to exocytotic sites on the β-cell surface, masking the antigenic exposure of ZnT8 and insulin after glucose-stimulated insulin secretion. In vivo administration of mAb43 to NOD mice selectively increased the proportion of regulatory T cells in the islet, resulting in complete and sustained protection against T1D onset as well as reversal of new-onset diabetes. The mAb43-induced self-tolerance was reversible after treatment cessation, and no adverse effects were exhibited during long-term monitoring. Our findings suggest that mAb43 masking of the antigenic exposure of β-cells suppresses the immunological cascade from B-cell antigen presentation to T cell–mediated β-cell destruction, providing a novel islet-targeted and antigen-specific immunotherapy to prevent and reverse clinical T1D.

Article Highlights
  • mAb43, a zinc transporter 8 (ZnT8)–specific monoclonal antibody, provides lasting protection against autoimmune diabetes in NOD mice.

  • The in vivo islet specificity of mAb43 enables targeted therapy to enhance safety.

  • High-affinity binding of mAb43 to the extracellular surface of ZnT8 shields β-cells from antigenic exposure.

  • β-Cell masking results in a localized increase in regulatory T cells in the pancreatic islet.

  • Prolonged mAb43 treatment clears destructive insulitis, preserves β-cell mass, and reverses seroconversion of insulin autoantibodies in NOD mice.

  • The immunological processes bridging the masking of specific cell-surface autoantigens and the broad suppression of polyspecific T-cell autoimmunity are still unclear.

During the preclinical phase of type 1 diabetes (T1D), four dominant autoantigens (insulin, GAD65, IA-2, and zinc transporter 8 [ZnT8]) are targeted by pathogenic lymphocytes (1). Cell-surface autoantigens and MHC-restricted T-cell epitopes are the first molecular markers that autoreactive lymphocytes encounter when recognizing β-cells. Because of unsuccessful attempts to immunolabel live human islet cells with autoreactive human sera in the past (2), cell-surface autoantigens have not been considered viable targets for immunotherapy. However, these early methods were designed to immunolabel static cell-surface autoantigens, whereas ZnT8, a dynamic autoantigen linked to the recycling of insulin secretory granules, can only be immunolabeled on the β-cell surface after glucose stimulation and endocytosis arrest (3,4). Our recent studies have detected ZnT8 autoantibodies targeting extracellular epitopes in patients with new-onset T1D (57), indicating that ZnT8 is a unique cell-surface marker for β-cell recognition by antigen-specific autoreactive B cells.

B cells are efficient antigen-presenting cells that rely on antigen uptake through the B-cell receptor (BCR) (8). ZnT8 has a Y-shaped dimeric structure similar to an immunoglobin (9), making it naturally prone to multivalent BCR ligation for ZnT8 uptake and processing at the B-cell synapse (10). B-cell antigen presentation is a crucial T1D-precipitating event in vivo; it involves cross-presentation of autoantigens to cognate CD8+ T cells (11) and promotes intraislet CD8+ cytotoxic T-cell survival (12). Masking ZnT8 from BCR recognition could directly block ZnT8 uptake by B cells. Furthermore, insulin and other secretory granule-derived autoantigens may be colocalized with ZnT8 at specific sites of exocytosis on the β-cell surface (13). They could be cocaptured with ZnT8 by autoreactive B cells as bystander antigens to induce polyspecific T-cell responses (14). Thus, binding of antibody and ZnT8 at the β-cell surface may mask multiple β-cell autoantigens, leading to a broader suppression of autoreactive B cells in the early stages of the immunological cascade before it gains enough momentum to precipitate cytotoxic T cells as the final mediator of β-cell destruction (15).

In the current study, we used a cell-surface ZnT8 antibody (mAb43) to mask β-cells in NOD mice, a model of spontaneous autoimmune diabetes. mAb43 is a ZnT8-boosted autoantibody that possesses a significantly stronger binding affinity to ZnT8 than natural ZnT8 autoantibodies by several orders of magnitude (4). The binding of mAb43 can effectively mask the entire extracellular surface of ZnT8, competitively obstructing the recognition of β-cells by polyclonal autoantibodies from patients with T1D and corresponding BCRs (4). Moreover, ZnT8 is a tissue-specific autoantigen expressed solely in the pancreatic islet (16,17). Systemically administered mAb43 results in specific mAb43 accumulation within pancreatic islets and persistent ZnT8 binding in vivo (4). However, the exact in vivo consequences of β-cell masking by mAb43 are still unknown. The current study evaluated the long-term efficacy and safety of repeated mAb43 dosing in NOD mice.

Animals

NOD/ShiLtJ (NOD), C57BL/6J (B6), and B6.BKS(D)-Leprdb/J (B6 db) mice were purchased from The Jackson Laboratory. A total of 201 NOD female mice were randomly distributed into various treatment cohorts. A small number of NOD mice were bred in house as detailed in Supplementary Fig. 1. All mouse procedures were approved by the Institutional Animal Care and Use Committee for animal welfare of Johns Hopkins University.

Cell Line

Human EndoC-β1H cells were cultured as described previously (18).

Antibody Production

Mouse mAb43 from a hybridoma clone was produced in an animal origin–free and serum-free medium and purified by size-exclusion high-performance liquid chromatography in PBS under sterile conditions. An isotype control antibody (mouse immunoglobulin G2b [IgG2b]) was purchased from Bioxcell and repurified as above. mAb43-mScalet was produced as described previously (4).

Immunofluorescence Labeling, Imaging, and Data Analysis

Live dispersed human pancreatic islet cells from a 42-year-old female donor (RRID: SAMN34130383) were stimulated with 20 mmol/L glucose at 37°C, chilled on ice, and then immunolabeled with mAb43 (1:50 = 20 μg/mL) and an Alexa 647–labeled secondary antibody (1:200), counterstained with DAPI, and imaged on a confocal microscope. To image the colocalization of ZnT8 and insulin on the β-cell surface, live EndoC-β1H cells in a monolayer cell culture were coimmunolabeled using an anti-insulin antibody (1:50) with mAb43 (1:50) or anti-CD71 (1:50), otherwise using an identical protocol as above. To examine the effect of mAb43 blockade, mAb43 (1:50) was added to the cell culture medium during glucose stimulation. Immunofluorescence was quantified using ImageJ. Immunofluorescence labeling of CD4+FoxP3+CD25+ regulatory T cells (Tregs) was performed on paraformaldehyde-fixed frozen sections of NOD pancreata.

mAb43 Uptake in Human Islet Cells

Human islets, isolated from nondiabetic cadaveric pancreata by Prodo Laboratories, were exposed to the mScarlet-tagged monoclonal antibody (mAb) in the islet culture medium up to 60 h and monitored over time for intraislet mScarlet fluorescence. Next, the islets were paraformaldehyde fixed, and the intracellularly trapped mAb was coimmunolabeled with insulin and glucagon for individual islet cell imaging in intact islets.

mAb43 Administration

mAb43 at 5 mg/kg, isotype-matched mouse IgG2b at 5 mg/kg, or PBS was administered weekly to female NOD mice by intravenous injections. The dosing schemes and numbers of mice in various treatment groups are detailed in Results. For new onset diabetic mice, mAb43 or isotype control was adminstered daily for 5 days followed by weekly dosing as described above.

Diabetes Monitoring

Mice were monitored weekly for 16-h fasting blood glucose (FBG) levels. Diabetes onset was defined by two consecutive FBG readings >250 mg/dL.

β-Cell Mass Measurements

Pancreata were paraformaldehyde fixed and paraffin embedded. Five pancreatic sections of 5-μm thickness across the mouse pancreas were immunohistochemically stained for insulin with an eosin counterstain. The percentage of the pancreas that was positive for insulin staining was measured on digital pathology images and then multiplied by pancreas weight to assess β-cell mass.

Insulitis Scoring

Histological assessment of insulitis was performed on hematoxylin-eosin–stained pancreatic sections on a scale of 0–3, such that 0 indicates no infiltrate; 1, peri-islet infiltrate; 2, mild intraislet infiltrate (<50%); and 3, severe intraislet infiltrate (>50%).

Immunophenotyping

Pancreatic islets, pancreatic lymph nodes (PLNs), and spleens were isolated, dispersed into single cells, and suspended at a concentration of 1 × 106 cells/mL. The single-cell suspension was then stimulated by phorbol-12-myristate-13-acetate (10 ng/mL) and ionomycin (250 ng/mL) followed by brefeldin (3.0 μg/mL) inhibition of cytokine secretion. Next, cells were sequentially incubated with Zombie NIR dye (1:200), Fc-γ receptor blocker (1:50), and monocyte/macrophage blocker (1:20) and then immunolabeled for cell-surface markers (1 μg antibody per million cells), followed by brief fixation and permeabilization before immunolabeling for intracellular FoxP3. The list of fluorophore-conjugated primary and isotype control antibodies is provided in the Supplementary Methods. Immunolabeled cells were acquired on an Aurora 4L spectral flow cytometer and analyzed using FlowJo. Reference spectra for individual fluorophores were generated using microspheres labeled with the same primary antibodies. The spectral data were unmixed, with dead cells excluded, and then gated for individual markers using isotype controls to set threshold levels for nonspecific immunolabeling. Cell populations expressing individual markers were calculated as overall percentages of total CD4+ or CD19+ cells or lymphocytes.

GSIS

Mouse islets were isolated and cultured as described previously (4). Size-matched islets were exposed to mAb43 (10 μg/mL), mAb20 (10 μg/mL), or mouse IgG2b (10 μg/mL) in the presence or absence of a cytokine cocktail consisting of mouse interleukin-1β (0.5 ng/mL), tumor necrosis factor-α (1 ng/mL), and interferon-γ (10 ng/mL). These islets were incubated for 24 h, and then basal insulin secretion, glucose-stimulated insulin secretion (GSIS) with 20 mmol/L glucose and 50 μmol/L IBMX, and total cellular insulin content were measured in triplicate using a homogeneous time-resolved fluorescence immunoassay (19).

Glucose Tolerance Testing

B6 db or NOD mice were fasted for 4 h, and blood glucose level was measured before and after peritoneal glucose injection (2 mg/kg) at 15, 30, 60, and 120 min. Sera were collected for insulin measurement before and after glucose injection at 0, 2, 8, 15, and/or 30 min.

Statistical Analysis

Data were analyzed using R (version 4.3.1) and are expressed as mean ± SE or median (interquartile range) for continuous variables or as count and percentage for categorical variables. P values ≤0.05 were considered statistically significant. R codes and outputs are available in the Supplementary Results.

Data and Resource Availability

All data generated during this study are included in the published article and its online supplementary files. Reagents generated are available upon reasonable request. Johns Hopkins University requires material transfer agreements for all inbound or outbound research materials.

Masking Antigenic Exposure of ZnT8 and Insulin

Glucose stimulation can induce the appearance of ZnT8 on the cell surface of human and rodent β-cell lines as well as primary mouse islet cells (3,4). Immunolabeling of live primary human islet cells with mAb43 also revealed discrete dots or granule-like clusters on the cell surface (Fig. 1A). Double immunolabeling of human EndoC-β1H cells with anti-insulin and mAb43 or anti-CD71 antibody demonstrated partial colocalization of insulin with ZnT8, but not with CD71 (Fig. 1B). In contrast, mAb20, a ZnT8-specific mAb targeting a cytosolic epitope (20), did not immunolabel ZnT8 on the β-cell surface, as its epitope was inaccessible to the extracellular mAb20 (Fig. 1B). Because CD71 is not linked to the insulin secretory pathway, its immunofluorescence was used as an independent in-cell reference to normalize the surface intensity of insulin immunolabeling (Fig. 1C). Notably, extracellular mAb43 significantly decreased the intensity of cell-surface insulin immunofluorescence compared with mAb20 (Fig. 1D), demonstrating that mAb43–ZnT8 binding masks ZnT8 on the β-cell surface and concomitantly reduces the antigenic exposure of insulin, which remains localized to exocytotic sites on the cell-surface membrane (13).

Figure 1

Cell-surface mAb43 binding reduces antigenic exposure of membrane-bound insulin. A: Immunolabeling of ZnT8 on live human islet cells with mAb43 (yellow). B: Coimmunolabeling of insulin (green) on the cell surface of live EndoC-β1H cells with mAb43 (red), anti-CD71 (red), or mAb20 (red) as indicated. Z-stack images spanning a 3.5-μm slab at 0.5-μm intervals are shown as maximum-intensity projection, with matched fluorescence intensities between the green and red channels. Manders’ colocalization coefficients (M1 and M2) quantify the overlap between green/red and red/green signals, respectively. C: EndoC-β1H cells stimulated with glucose in the presence of mAb43 or mAb20 as indicated and then coimmunolabeled on ice for insulin (green) and CD71 (yellow). Z-stack images are displayed without intensity matching to reveal the actual insulin fluorescence intensity compared with that of CD71. D: Normalized cell-surface insulin fluorescence intensities in individual EndoC-β1H cells, with error bars representing SE. E: mScarlet (a red fluorescent protein) uptake in live human islets exposed to mScarlet-mAb43 or mScarlet-mAb20 as indicated. Maximum-intensity projections of Z-stack images of islets are shown. F: mScarlet-mAb43 uptake in individual islet cells coimmunolabeled for mAb43 (red), insulin (green), and glucagon (yellow) as indicated. Arrowheads and arrows indicate α- and β-cells, respectively. ***P < 0.01 by two-tailed Welch t test for two independent means.

Figure 1

Cell-surface mAb43 binding reduces antigenic exposure of membrane-bound insulin. A: Immunolabeling of ZnT8 on live human islet cells with mAb43 (yellow). B: Coimmunolabeling of insulin (green) on the cell surface of live EndoC-β1H cells with mAb43 (red), anti-CD71 (red), or mAb20 (red) as indicated. Z-stack images spanning a 3.5-μm slab at 0.5-μm intervals are shown as maximum-intensity projection, with matched fluorescence intensities between the green and red channels. Manders’ colocalization coefficients (M1 and M2) quantify the overlap between green/red and red/green signals, respectively. C: EndoC-β1H cells stimulated with glucose in the presence of mAb43 or mAb20 as indicated and then coimmunolabeled on ice for insulin (green) and CD71 (yellow). Z-stack images are displayed without intensity matching to reveal the actual insulin fluorescence intensity compared with that of CD71. D: Normalized cell-surface insulin fluorescence intensities in individual EndoC-β1H cells, with error bars representing SE. E: mScarlet (a red fluorescent protein) uptake in live human islets exposed to mScarlet-mAb43 or mScarlet-mAb20 as indicated. Maximum-intensity projections of Z-stack images of islets are shown. F: mScarlet-mAb43 uptake in individual islet cells coimmunolabeled for mAb43 (red), insulin (green), and glucagon (yellow) as indicated. Arrowheads and arrows indicate α- and β-cells, respectively. ***P < 0.01 by two-tailed Welch t test for two independent means.

Close modal

mAb43 Uptake in Human Islet Cells

Cell surface–bound mAb43 underwent rapid internalization at 37°C. A comparison of live human islets exposed to mScarlet-tagged mAb43 and mAb20 demonstrated selective islet uptake of mAb43-mScarlet over mAb20-mScarlet, indicating receptor-mediated mAb43 endocytosis (Fig. 1E). The intracellularly trapped mAb43-mScarlet was confirmed by anti-IgG immunolabeling of paraformaldehyde-fixed islets. Coimmunolabeling for insulin and glucagon revealed the presence of mAb43-mScarlet in both α- and β-cells (Fig. 1F).

mAb43 Treatment Prevents Spontaneous T1D in NOD Mice

Weekly mAb43 administration to mice at a dose of 5 mg/kg can maintain β-cell masking with no detectable off-target biodistribution (4). In the current study, weekly mAb43 injection was initiated at 10 weeks of age, and FBG levels were monitored weekly to track disease development (Fig. 2A). NOD mice receiving either isotype-matched IgG2b or PBS vehicle showed no differences in glycemic profile (Fig. 2B) or diabetes incidence (P = 1.0 by Kaplan-Meier survival analysis with log-rank test). Consequently, mice from the isotype and PBS groups were combined into a single control group. A total of 64 female NOD mice were assigned to the mAb43 treatment group, whereas the control group consisted of 40 female mice. Subsets of mice from each group were randomly selected for terminal assessments at 20, 25, 30, and 35 weeks of age, as well as for evaluation of treatment reversal after discontinuing mAb43 injections at different time points (Fig. 3A). Throughout the study to 35 weeks of age, all mice in the mAb43 treatment group maintained euglycemia, except for one mouse that experienced a hyperglycemic episode from weeks 21 to 25 (Fig. 2C). However, this mouse was still classified as nondiabetic because its FBG subsequently returned to <250 mg/dL (Fig. 2A). In contrast, all mice in the control group developed diabetes by 35 weeks of age (Fig. 2C), underscoring the complete protection provided by mAb43 treatment.

Figure 2

mAb43 prevents diabetes onset. A: FBG levels over time from individual mice treated with mAb43 from 10 to 35 weeks. Numbers of mice after removal for terminal analyses are indicated at different time points. Dashed gray line represents the 250-mg/dL FBG threshold for diagnosis of diabetes. B: FBG levels in the control group receiving weekly isotype (red) or PBS (magenta) injections as indicated, within the same treatment window as in A. Number of mice remaining at each time point is shown. C: Percentages of nondiabetic mice in the treatment (blue) or combined control (red) group from 10 to 35 weeks of age as indicated. D: FBG levels over time during the mAb43 treatment window from 14 to 40 weeks. Number of mice is indicated. E: FBG levels in the control group receiving weekly isotype (red) or PBS (magenta) injections as indicated within the same time window as in D. F: Percentages of nondiabetic mice in the treatment (blue) or combined control (red) group from 14 to 40 weeks of age as indicated. ***P < 0.01 by Kaplan-Meier survival analysis with log-rank test.

Figure 2

mAb43 prevents diabetes onset. A: FBG levels over time from individual mice treated with mAb43 from 10 to 35 weeks. Numbers of mice after removal for terminal analyses are indicated at different time points. Dashed gray line represents the 250-mg/dL FBG threshold for diagnosis of diabetes. B: FBG levels in the control group receiving weekly isotype (red) or PBS (magenta) injections as indicated, within the same treatment window as in A. Number of mice remaining at each time point is shown. C: Percentages of nondiabetic mice in the treatment (blue) or combined control (red) group from 10 to 35 weeks of age as indicated. D: FBG levels over time during the mAb43 treatment window from 14 to 40 weeks. Number of mice is indicated. E: FBG levels in the control group receiving weekly isotype (red) or PBS (magenta) injections as indicated within the same time window as in D. F: Percentages of nondiabetic mice in the treatment (blue) or combined control (red) group from 14 to 40 weeks of age as indicated. ***P < 0.01 by Kaplan-Meier survival analysis with log-rank test.

Close modal
Figure 3

Loss of protection after treatment cessation. A: FBG levels over time in mAb43-treated mice after discontinuing weekly mAb43 injections at 20, 25, 30, 35, or 40 weeks of age as indicated by arrows. Glucose monitoring was continued until the onset of diabetes. Number of mice in each group is shown. B: Percentages of nondiabetic mice over time after discontinuing weekly mAb43 injections as described in A.

Figure 3

Loss of protection after treatment cessation. A: FBG levels over time in mAb43-treated mice after discontinuing weekly mAb43 injections at 20, 25, 30, 35, or 40 weeks of age as indicated by arrows. Glucose monitoring was continued until the onset of diabetes. Number of mice in each group is shown. B: Percentages of nondiabetic mice over time after discontinuing weekly mAb43 injections as described in A.

Close modal

Sustained T1D Protection by Continuous mAb43 Dosing

We continuously monitored five NOD mice to assess long-term mAb43 protection with a delayed treatment initiation at 14 weeks of age (Fig. 2D). This later intervention was inadequate to prevent the onset of acute T1D in one of the five mice. However, the remaining four mice maintained euglycemia until 40 weeks of age, whereas all mice in the control group (n = 13) developed diabetes (Fig. 2E and F). After treatment cessation at 40 weeks of age, all four mAb43-treated mice remained nondiabetic until 75 weeks of age (Fig. 3A right). These findings suggest that mAb43 treatment provided prolonged but not indefinite protection after treatment cessation.

Reversal of mAb43 Protection After Treatment Cessation

To further assess the reversibility of mAb43 therapy, we continuously monitored FBG after discontinuation of mAb43 treatment at 20, 25, 30, or 35 weeks of age (Fig. 3A). Over time, all mice gradually developed diabetes, with varying lag times to disease onset (Fig. 3B). The complete reversal of the mAb43 protective effect indicated a causal relationship between mAb43 treatment and diabetes prevention.

mAb43 Treatment Preserves β-Cell Mass

We conducted immunohistochemistry quantification of insulin staining on cross-sections of NOD pancreata from mice in the treatment and control groups at different time points, with or without diabetes (Fig. 4A). All mAb43-treated mice remained nondiabetic and displayed significantly higher levels of β-cell mass compared with diabetic mice at all ages.

Figure 4

Continuous mAb43 dosing preserves β-cell mass and reduces insulitis. A: Representative images of pancreatic islets revealed by anti-insulin immunohistochemistry. Shown are 20-week-old diabetic (D) or nondiabetic (ND) mice from the control group or ND mice from the mAb43 treatment group. The right panel displays changes in islet mass for mice in different treatment groups, ages, and D statuses as indicated. Boxes represent the statistically central range of data from the boundaries of upper 25% to lower 25% quartiles. Red and black lines indicate the median and average values of the data, respectively. Number of mice in each group and their significance levels compared with 20-week-old D mice in the control group are indicated. B: Representative hematoxylin-eosin–stained pancreatic sections obtained from 20-week-old ND NOD mice in the control group and their associated insulitis grading as indicated. Changes in percentages of four insulitis categories are summarized in stacked histograms (right) for mice in different treatment groups, ages, and D statuses as indicated. Data are from 157 to 618 islets in 3–10 mice as shown in A. C: Schematic of radioimmunoassay for measuring the serum IAA level and the associated results (right panel) obtained from 3–10 mice in different treatment groups, ages, and D statuses as indicated. Dashed gray line indicates the IAA positivity threshold, and the percentages of IAA+ or IAA mice are shown in red or blue, respectively. *P < 0.05, ***P < 0.01 by Kruskal-Wallis analysis with post hoc Mann-Whitney U test for pairwise comparison of the medians (A) or by χ2 analysis with post hoc multiple comparisons with the D control at 20 weeks of age (B).

Figure 4

Continuous mAb43 dosing preserves β-cell mass and reduces insulitis. A: Representative images of pancreatic islets revealed by anti-insulin immunohistochemistry. Shown are 20-week-old diabetic (D) or nondiabetic (ND) mice from the control group or ND mice from the mAb43 treatment group. The right panel displays changes in islet mass for mice in different treatment groups, ages, and D statuses as indicated. Boxes represent the statistically central range of data from the boundaries of upper 25% to lower 25% quartiles. Red and black lines indicate the median and average values of the data, respectively. Number of mice in each group and their significance levels compared with 20-week-old D mice in the control group are indicated. B: Representative hematoxylin-eosin–stained pancreatic sections obtained from 20-week-old ND NOD mice in the control group and their associated insulitis grading as indicated. Changes in percentages of four insulitis categories are summarized in stacked histograms (right) for mice in different treatment groups, ages, and D statuses as indicated. Data are from 157 to 618 islets in 3–10 mice as shown in A. C: Schematic of radioimmunoassay for measuring the serum IAA level and the associated results (right panel) obtained from 3–10 mice in different treatment groups, ages, and D statuses as indicated. Dashed gray line indicates the IAA positivity threshold, and the percentages of IAA+ or IAA mice are shown in red or blue, respectively. *P < 0.05, ***P < 0.01 by Kruskal-Wallis analysis with post hoc Mann-Whitney U test for pairwise comparison of the medians (A) or by χ2 analysis with post hoc multiple comparisons with the D control at 20 weeks of age (B).

Close modal

Prolonged mAb43 Treatment Reduces Insulitis

The extent of insulitis reflects autoimmune progression. Grading hematoxylin-eosin–stained pancreatic sections revealed two distinct patterns of lymphocytic infiltration in 20-week-old diabetic and nondiabetic mice in the control group (Fig. 4B). The higher or lower levels of insulitis severity corresponded to NOD subtypes with acute or progressive T1D onset (21). The age-matched mAb43-treated group contained both subtypes and thus exhibited median insulitis severity between that observed in controls with acute or progressive onset (Fig. 4B). Notably, extending mAb43 treatment from 20 to 30 weeks progressively reduced insulitis severity, culminating in a significant clearance of destructive infiltrates by 35 weeks of age (Fig. 4B). Moreover, mice with later diabetes onset at 42–47 and 75 weeks of age had an insulitis rebound (Fig. 4B); however, insulitis in these diabetic mice was less severe and associated with higher residual β-cell mass levels compared with that in control mice with diabetes at 20 weeks of age (Fig. 4A).

Seroreversion of IAAs

Serum insulin autoantibodies (IAAs) are serological biomarkers for insulin-specific autoimmunity. Consistent with a significant clearance of destructive insulitis at 35 weeks of age (Fig. 4B), the same nondiabetic mice exhibited complete IAA negativity (Fig. 4C). This shift to IAA negativity in conjunction with the preservation of β-cell mass (Fig. 4A) is considered a reliable predictor for the induction of insulin-specific immune tolerance in NOD mice (22). Nonetheless, the establishment of this immune tolerance was only temporary, being lost after mAb43 treatment cessation (Fig. 3B). Furthermore, all mice with established diabetes were IAA negative (Fig. 4C), indicative of a significant reduction in insulin secretion that led to a decrease in the stimulation of the insulin-specific B-cells to produce IAAs.

Reversal of New-Onset T1D

We further examined the efficacy of mAb43 therapy in two different diabetic states. First, we examined diabetes reversal in newly diagnosed NOD mice during later stages of adulthood. Accordingly, we delayed diabetes onset with mAb43 treatments from 10 weeks of age followed by discontinuation of mAb43 treatments between 20 to 35 weeks of age (Fig. 3A) and then resumed mAb43 or isotype treatment within 48 h of diabetes onset. There was an average 13-week interval between the cessation of mAb43 pretreatment and the initiation of mAb43 retreatment (Fig. 3B). Given the short half-life of mAb43 pancreatic retention (6 days) (4), mAb43 carryover from the pretreatment was negligible. To avoid the confounding effect of insulin on β-cell survival (23), exogenous insulin was not administered to diabetic mice. All mice receiving mAb43 exhibited a progressive decline in FBG, which eventually stabilized at various levels (Fig. 5A). Nine of 10 mice achieved remission, with one remaining mildly diabetic. In contrast, all control group mice remained severely diabetic until their death (Fig. 5B). Kaplan-Meier survival analysis showed a significant increase in survival compared with the control group (Fig. 5C). Next, we investigated diabetes reversal in young NOD mice without mAb43 pretreatment (Fig. 5D–F). Despite differing ages at diabetes onset, isotype treatments revealed no significant difference in postonset survival between diabetic mice with and without mAb43 pretreatment (P = 0.6 by Kaplan-Meier survival analysis with log-rank test). This absence of significant effects on postonset disease progression was consistent with the inability of mAb43 pretreatment to establish permanent immune tolerance (Fig. 3B). All young diabetic mice experienced a gradual reduction in FBG levels with mAb43 treatment, but this fell short of an achievement of remission before death (Fig. 5D). Nevertheless, mAb43 treatment significantly extended postonset survival compared with isotype treatment in the control group (Fig. 5F). These findings suggest mAb43 therapy is more effective in reversing delayed autoimmune diabetes in older mice.

Figure 5

Diabetes reversal induced by mAb43 therapy. A: Changes in FBG levels over time in NOD mice with delayed diabetes onset induced by mAb43 pretreatment. mAb43 therapy commenced within 48 h of diabetes onset and continued until death or terminal analysis at 12 weeks postonset. Inset shows steady-state FBG levels for individual mice. B: Changes in FBG levels over time for littermate diabetic NOD mice receiving isotype control injections until death. Ages at diabetes onset in the mAb43 treatment and control groups were 40.7 ± 1.5 and 33.1 ± 3.0 weeks, respectively. C: Survival percentages over time for diabetic mice described in A and B in the mAb43 treatment group (blue) and control group (red). D: Changes in FBG levels over time for mAb43-treated mice that had developed spontaneous diabetes without mAb43 pretreatment. E: Changes in FBG levels over time for isotype-treated littermate diabetic NOD mice. F: Survival percentages over time for mAb43- or isotype-treated mice described in D and E. Average ages at diabetes onset in the mAb43 treatment (blue) and control (red) groups were 22.9 ± 0.8 and 21.2 ± 1.0 weeks, respectively. G: Insulin immunohistochemistry (IHC) or hematoxylin-eosin (H&E) staining of pancreas sections from diabetic NOD mice with isotype treatment at 3 weeks postonset (upper) or with mAb43 treatment at 12 weeks postonset (lower). Arrows indicate residual islets with or without insulin staining. H: Detection of proliferating β-cells in remitted NOD mice at 12 weeks postonset by immunofluorescence costaining of pancreatic sections for insulin, Ki67, and DAPI. Arrows indicate Ki67-positive β-cells in the islets. I: Residual islet mass in diabetic NOD mice at 3 weeks postonset with isotype treatment or 12 weeks postonset with mAb43 treatment as indicated. J: Percentages of islets in four insulitis categories for diabetic NOD mice at 3 weeks postonset with isotype treatment or 12 weeks postonset with mAb43 treatment. K: Glucose tolerance tests (GTTs) in mAb43-treated NOD mice at 12 weeks postonset (blue) compared with those in new-onset NOD mice with isotype treatment (red). Note that GTTs were performed in the same mAb43-treated mice used for survival and terminal analyses in A and GJ. ***P < 0.01 by Kaplan-Meier analysis with log-rank test (C), by Kruskal-Wallis analysis with post hoc Mann-Whitney U test for pairwise comparison of the medians (I), or by χ2 analysis with post hoc multiple comparisons (J).

Figure 5

Diabetes reversal induced by mAb43 therapy. A: Changes in FBG levels over time in NOD mice with delayed diabetes onset induced by mAb43 pretreatment. mAb43 therapy commenced within 48 h of diabetes onset and continued until death or terminal analysis at 12 weeks postonset. Inset shows steady-state FBG levels for individual mice. B: Changes in FBG levels over time for littermate diabetic NOD mice receiving isotype control injections until death. Ages at diabetes onset in the mAb43 treatment and control groups were 40.7 ± 1.5 and 33.1 ± 3.0 weeks, respectively. C: Survival percentages over time for diabetic mice described in A and B in the mAb43 treatment group (blue) and control group (red). D: Changes in FBG levels over time for mAb43-treated mice that had developed spontaneous diabetes without mAb43 pretreatment. E: Changes in FBG levels over time for isotype-treated littermate diabetic NOD mice. F: Survival percentages over time for mAb43- or isotype-treated mice described in D and E. Average ages at diabetes onset in the mAb43 treatment (blue) and control (red) groups were 22.9 ± 0.8 and 21.2 ± 1.0 weeks, respectively. G: Insulin immunohistochemistry (IHC) or hematoxylin-eosin (H&E) staining of pancreas sections from diabetic NOD mice with isotype treatment at 3 weeks postonset (upper) or with mAb43 treatment at 12 weeks postonset (lower). Arrows indicate residual islets with or without insulin staining. H: Detection of proliferating β-cells in remitted NOD mice at 12 weeks postonset by immunofluorescence costaining of pancreatic sections for insulin, Ki67, and DAPI. Arrows indicate Ki67-positive β-cells in the islets. I: Residual islet mass in diabetic NOD mice at 3 weeks postonset with isotype treatment or 12 weeks postonset with mAb43 treatment as indicated. J: Percentages of islets in four insulitis categories for diabetic NOD mice at 3 weeks postonset with isotype treatment or 12 weeks postonset with mAb43 treatment. K: Glucose tolerance tests (GTTs) in mAb43-treated NOD mice at 12 weeks postonset (blue) compared with those in new-onset NOD mice with isotype treatment (red). Note that GTTs were performed in the same mAb43-treated mice used for survival and terminal analyses in A and GJ. ***P < 0.01 by Kaplan-Meier analysis with log-rank test (C), by Kruskal-Wallis analysis with post hoc Mann-Whitney U test for pairwise comparison of the medians (I), or by χ2 analysis with post hoc multiple comparisons (J).

Close modal

Histological Characterization of Diabetes Reversal

All diabetic mice, regardless of mAb43 treatment, displayed significantly reduced islet mass compared with nondiabetic mice (Fig. 5I vs. Fig. 4A). In isotype-treated mice, most remaining islets at 3 weeks postonset lacked insulin immunostaining, whereas mice treated with mAb43 and in remission retained a substantial proportion of insulin-positive islets at 12 weeks postonset (Fig. 5G). This preservation of β-cell mass resulted in a noteworthy improvement in blood glucose clearance compared with that in isotype-treated diabetic mice (Fig. 5K). Ki67 immunolabeling revealed proliferating β-cells within the islets (Fig. 5H), although their low frequency suggested minimal contribution to islet expansion. The expanded insulin-positive areas in Fig. 5G were a result of surrounding immune cells that internalized secreted insulin from β-cells. Insulitis persisted in all mice examined, but mAb43 treatment promoted nondestructive peri-islet infiltrates (Fig. 5J). These findings indicate that mAb43 therapy preserves residual β-cell mass and function in the presence of ongoing insulitis.

Islet-Resident Treg Induction

The immunoregulatory effects of mAb43 on insulitis were investigated ex vivo using pancreatic sections from 20-week-old nondiabetic mice that received weekly injections of either mAb43 or isotype control, starting at 10 weeks of age (n = 2 × 10). Anti-CD4, FoxP3, and CD25 coimmunolabeling revealed no significant differences in the numbers of islet-residing CD4+ T cells between the mAb43 and control groups (Fig. 6A and B). However, mAb43 treatment significantly increased the number of CD4+FoxP3+CD25+ T cells within the islets (Fig. 6B). Notably, FoxP3 and CD25 immunofluorescence exhibited a high degree of colocalization in all FoxP3+ islets. As a result, CD4+FoxP3+CD25 T cells were not detected (Fig. 6B). Thus, mAb43 treatment increased the Treg population within islet infiltrates.

Figure 6

Selective enhancement of tolerogenic phenotypes of islet infiltrates by mAb43 treatment. A: Immunofluorescence colabeling of representative pancreatic sections from 20-week-old nondiabetic NOD mice for CD4 (red), Foxp3 (green), and CD25 (yellow). Islet boundary is outlined by a dashed circle, and islet-resident CD4+ T cells are either FoxP3CD25 (upper) or FoxP3+CD25+ (middle) with single-cell resolution as indicated by arrows in the lower panel. B: Quantification of islet-resident CD4+ T cells, CD4+FoxP3+CD25+ Tregs, and CD4+FoxP3+CD25 non-Tregs by enumerating individual islet-resident CD4+ T cells positively or negatively immunolabeled for FoxP3 and CD25 in pancreatic sections from 20-week-old nondiabetic NOD mice with mAb43 treatment (blue; n = 10 mice) and age-matched nondiabetic NOD mice with isotype treatment (red; n = 10 mice). A total of 120 islets from each treatment group were examined. CH: Immunophenotyping of tissue-resident lymphocytes isolated from the pancreatic islets, PLNs, and spleen of 20-week-old nondiabetic NOD mice in the mAb43 (blue) or isotype (red) treatment groups. A total of seven mice in each group were examined. Cell populations expressing individual markers were calculated as percentages per total lymphocyte or CD19+ or CD4+ population as indicated. Shown are overall percentages of CD19+ B cells, CD4+ and CD8+ T cells, and CD19+ B cells expressing CD40, CD80, and MHC class II. I: Representative histograms showing the distribution of FoxP3 expressing cells in CD4+ populations from the islets, PLNs, and spleens of a pair of mice with mAb43 (blue) or isotype (red) treatment as indicated. The vertical dashed line marks the gates for CD4+FoxP3+ populations. J: Overall percentages of CD4+ T cells expressing FoxP3 in the islets, PLNs, and spleens as indicated. K: Schematic model for the mechanism of action. ZnT8 binds to BCR to activate the B cell, leading to cytotoxic effector T-cell activation. L: mAb43 masks the β-cell from BCR recognition, leading to an increase in the Treg cell proportion in the islet. ***P < 0.01 by two-tailed t test for two independent means (B or J).

Figure 6

Selective enhancement of tolerogenic phenotypes of islet infiltrates by mAb43 treatment. A: Immunofluorescence colabeling of representative pancreatic sections from 20-week-old nondiabetic NOD mice for CD4 (red), Foxp3 (green), and CD25 (yellow). Islet boundary is outlined by a dashed circle, and islet-resident CD4+ T cells are either FoxP3CD25 (upper) or FoxP3+CD25+ (middle) with single-cell resolution as indicated by arrows in the lower panel. B: Quantification of islet-resident CD4+ T cells, CD4+FoxP3+CD25+ Tregs, and CD4+FoxP3+CD25 non-Tregs by enumerating individual islet-resident CD4+ T cells positively or negatively immunolabeled for FoxP3 and CD25 in pancreatic sections from 20-week-old nondiabetic NOD mice with mAb43 treatment (blue; n = 10 mice) and age-matched nondiabetic NOD mice with isotype treatment (red; n = 10 mice). A total of 120 islets from each treatment group were examined. CH: Immunophenotyping of tissue-resident lymphocytes isolated from the pancreatic islets, PLNs, and spleen of 20-week-old nondiabetic NOD mice in the mAb43 (blue) or isotype (red) treatment groups. A total of seven mice in each group were examined. Cell populations expressing individual markers were calculated as percentages per total lymphocyte or CD19+ or CD4+ population as indicated. Shown are overall percentages of CD19+ B cells, CD4+ and CD8+ T cells, and CD19+ B cells expressing CD40, CD80, and MHC class II. I: Representative histograms showing the distribution of FoxP3 expressing cells in CD4+ populations from the islets, PLNs, and spleens of a pair of mice with mAb43 (blue) or isotype (red) treatment as indicated. The vertical dashed line marks the gates for CD4+FoxP3+ populations. J: Overall percentages of CD4+ T cells expressing FoxP3 in the islets, PLNs, and spleens as indicated. K: Schematic model for the mechanism of action. ZnT8 binds to BCR to activate the B cell, leading to cytotoxic effector T-cell activation. L: mAb43 masks the β-cell from BCR recognition, leading to an increase in the Treg cell proportion in the islet. ***P < 0.01 by two-tailed t test for two independent means (B or J).

Close modal

Islet-Localized Tolerogenic Immune Responses

The broader immunoregulatory effects of mAb43 were further examined using high-dimensional multiplexing flow cytometry to phenotype tissue-resident lymphocytes isolated from 20-week-old nondiabetic mice subjected to mAb43 or isotype treatment from 10 weeks of age (n = 2 × 7). mAb43 did not significantly alter the percentages of CD19+ B cells, CD4+ T cells, or CD8+ T cells within the lymphocyte populations residing in islets, PLNs, or spleens (Fig. 6C–E). Similarly, mAb43 treatment had no significant impact on the percentages of CD19+CD40+, CD19+CD80+, or CD19+MHCII+ cells in these tissues (Fig. 6F–H). Although major immune cell subsets and their phenotypes remained largely unaffected, mAb43 treatment resulted in a shift in the islet-residing CD4+ T cells toward higher FoxP3 expression levels (Fig. 6I), leading to a significant increase in the percentage of CD4+FoxP3+ T cells (Fig. 6J). In contrast, the percentage of CD4+FoxP3+ T cells in the PLNs and spleen remained unchanged (Fig. 6I and J). Therefore, the immunophenotypic changes induced by mAb43 were primarily confined to the CD4+FoxP3+ pool in pancreatic islets, shifting the islet lymphocyte infiltrates toward a tolerogenic phenotype.

Lack of Detectable mAb43 Effects on β-Cell Functions

To investigate the direct impact of mAb43 therapy on the insulin secretory function of β-cells, we assessed insulin secretion and production using isolated islets from B6 mice, which were free from the confounding effects of insulitis. Although proinflammatory cytokine exposure increased basal insulin secretion regardless of mAb43 treatment (Fig. 7A), mAb43 alone or in combination with cytokines did not induce significant differences in GSIS or total insulin content within the islets (Fig. 7B and C). This observation is supported by data from young NOD mice before developing overt insulitis, where mAb43 injections from 3 to 6 weeks of age did not affect in vivo GSIS compared with isotype-treated mice (Fig. 7D). In contrast, mAb43 treatment of adult diabetic NOD mice preserved in vivo insulin secretion against persistent insulitis (Fig. 7E). Moreover, in an obesity-induced diabetes mouse model, weekly administration of mAb43 or isotype control to male B6 db mice from 6 to 17 weeks of age did not induce any signs of insulitis (Fig. 7F) or antibody-dependent cellular cytotoxicity, as evidenced by the absence of significant differences in FBG levels (Fig. 7G) and glucose tolerance tests at 10 and 15 weeks of age (Fig. 7H and I). In summary, our findings indicate that mAb43 therapy preserves β-cell function in the face of insulitis. The absence of antibody-dependent cellular cytotoxicity may be attributed to rapid internalization of surface-bound mAb43 in vivo.

Figure 7

Direct impact of mAb43 on insulin secretion and biosynthesis. A: Basal ex vivo insulin secretion from isolated mouse islets in Krebs buffer with 2 mmol/L glucose in the presence or absence of a proinflammatory cytokine cocktail. mAb43, mAb20, or mouse IgG isotype control was added to the culture overnight as indicated. Error bars indicate SE. B: GSIS from isolated mouse islets in Krebs buffer with 20 mmol/L glucose and 50 μmol/L IBMX in the presence or absence of cytokine as indicated. C: Total islet insulin content from isolated mouse islets. D: Serum insulin levels over time in 6-week-old NOD mice in response to a glucose injection. These mice received weekly injections of mAb43 (blue) or isotype (red) starting from 3 weeks of age. E: Serum insulin levels over time in response to a glucose injection to diabetic NOD mice in remission after mAb43 treatment at 12 weeks postonset (blue) or a glucose injection to diabetic NOD mice with isotype treatment at 3 weeks postonset (red). The same remitted mice were used in diabetes reversal experiments as described in Fig. 5A. F: Absence of insulitis in 17-week-old hyperglycemic male B6 db mice administered weekly mAb43 or isotype control injections from 6 to 17 weeks of age as indicated. G: FBG levels over time in male B6 db mice administered weekly mAb43 (blue) or isotype control (red) injections from 6 to 17 weeks of age (n = 2 × 5). H: Glucose tolerance tests (GTTs) in male B6 db mice at 10 weeks of age. Same mice were used in G. I: GTTs in male B6 db mice at 15 weeks of age. No statistically significant differences between treatment groups as determined by two-way ANOVA with repeated measures. ***P < 0.01 by two-tailed t test for two independent means.

Figure 7

Direct impact of mAb43 on insulin secretion and biosynthesis. A: Basal ex vivo insulin secretion from isolated mouse islets in Krebs buffer with 2 mmol/L glucose in the presence or absence of a proinflammatory cytokine cocktail. mAb43, mAb20, or mouse IgG isotype control was added to the culture overnight as indicated. Error bars indicate SE. B: GSIS from isolated mouse islets in Krebs buffer with 20 mmol/L glucose and 50 μmol/L IBMX in the presence or absence of cytokine as indicated. C: Total islet insulin content from isolated mouse islets. D: Serum insulin levels over time in 6-week-old NOD mice in response to a glucose injection. These mice received weekly injections of mAb43 (blue) or isotype (red) starting from 3 weeks of age. E: Serum insulin levels over time in response to a glucose injection to diabetic NOD mice in remission after mAb43 treatment at 12 weeks postonset (blue) or a glucose injection to diabetic NOD mice with isotype treatment at 3 weeks postonset (red). The same remitted mice were used in diabetes reversal experiments as described in Fig. 5A. F: Absence of insulitis in 17-week-old hyperglycemic male B6 db mice administered weekly mAb43 or isotype control injections from 6 to 17 weeks of age as indicated. G: FBG levels over time in male B6 db mice administered weekly mAb43 (blue) or isotype control (red) injections from 6 to 17 weeks of age (n = 2 × 5). H: Glucose tolerance tests (GTTs) in male B6 db mice at 10 weeks of age. Same mice were used in G. I: GTTs in male B6 db mice at 15 weeks of age. No statistically significant differences between treatment groups as determined by two-way ANOVA with repeated measures. ***P < 0.01 by two-tailed t test for two independent means.

Close modal

The experiments described herein demonstrate the efficacy of mAb43 in preventing and reversing new-onset T1D in NOD mice. These findings suggest that mAb43 acts on critical steps in autoimmune responses leading to T cell–mediated destruction of β-cells. In this process, autoantigens on the β-cell surface could act as molecular attractants, driving antigen-specific homing of autoreactive B cells to the islets. Infiltrating B cells can then acquire cell-surface autoantigens, generating T-cell epitopes for presentation to autoreactive T cells, thereby directing their homing to the islets and differentiation into cytotoxic effectors (24). Of note, autoreactive B cells are present in a substantial proportion of the circulating B-cell population in healthy individuals (25). ZnT8 is a major membrane-bound autoantigen (26). Surfaced ZnT8 may facilitate BCR cross-linking to gather ZnT8 into the B-cell synapse, leading to efficient ZnT8 uptake and processing and presentation of ZnT8-derived T-cell epitopes (Fig. 6K). Indeed, a ZnT8186–194 peptide is one of the dominant T-cell epitopes driving islet homing of pathogenic CD8+ T cells in human T1D (27).

The mechanism of mAb43 actions involves masking ZnT8 from B-cell recognition (Fig. 6L), preserving the pool of naive ZnT8-reactive B cells that can interact with cognate CD4+ T cells to generate inducible Tregs in the islet (28). This notion is in line with an increased local Treg number within the islet of mAb43-treated NOD mice (Fig. 6A and B). Tregs play a crucial role in suppressing cytotoxic CD8+ T-cell activation and promoting the regulatory milieu by secreting proinflammatory interleukin-10 (29). Recently, the US Food and Drug Administration approved the first-ever T1D-modifying therapy, teplizumab, an anti-CD3 mAb that suppresses immune responses through transient depletion of T cells and modulation of the CD3/T-cell receptor complex (30). In NOD mice, a side-by-side comparison between teplizumab and mAb43 revealed that both therapies effectively reverse new-onset T1D (31), but only mAb43 exhibited a safety profile applicable to chronic use.

Discontinuing mAb43 treatment caused the halted autoimmunity progression toward T1D to resume, even in elderly NOD mice (Fig. 3). This finding underscores the necessity for ongoing mAb43 treatment to achieve sustained efficacy. The chronic use of mAb43 may alter ZnT8-mediated zinc accumulation in the insulin secretory granule, where zinc is cocrystallized with insulin to form the insulin-dense core (32). Our data show that exposing mouse islets to mAb43 does not alter GSIS or islet insulin content ex vivo (Fig. 7). Previous work has demonstrated that ZnT8-knockout mice maintain normal insulin biosynthesis and GSIS despite the absence of zinc accumulation in insulin secretory granules (33). Additionally, ZnT8 haploinsufficiency in NOD mice protects against β-cell dysfunction by increasing mitochondrial respiration (34). Furthermore, downregulation of ZnT8 expression in human EndoC-β1H cells provides protection against inflammatory stress by reducing the burden of unfolded protein response to ZnT8 misfolding (19). In humans, ZnT8 haploinsufficiency has been associated with protection against type 2 diabetes, without adverse effects (35,36). The lifelong protective effects of ZnT8 downregulation demonstrate the safety of sustained ZnT8-suppressive therapy in humans (37). These findings suggest that ZnT8-specific mAb43 therapy is a viable long-term solution to prevent or reverse new-onset T1D.

T1D in humans exhibits clinical heterogeneity associated with genetic variations, diverse autoimmune responses, and environmental influences. Previous preclinical findings from inbred NOD mice have shown limited success when translated into clinical trials (38). However, mAb43 therapy stands out as a categorically distinct modality from all established lymphocyte-targeted immunotherapies. Unlike other approaches, mAb43 does not directly modify lymphocytes but rather preserves β-cells in the presence of insulitis. mAb43 binds equally to human and mouse ZnT8 on highly conserved and membrane-flush cell-surface epitopes (4), ensuring the preservation of mAb43-mediated β-cell masking regardless of differences between human and mouse immune systems. Importantly, ZnT8 is a major target of pathogenic CD8+ T cells in human T1D (27,39), further supporting the clinical relevance of ZnT8-masking therapy. Islet autoantibodies maternally transferred to the fetus seem to be protective against future islet autoimmunity in children born to mothers with T1D (4042). This protective role of human islet autoantibodies is mirrored by the protective effects of mAb43, functioning as a cell-surface autoantibody to provide long-term β-cell protection without adverse effects.

Acknowledgments. The authors thank Xiaoling Zhang from the Ross Flow Cytometry Core, Johns Hopkins University School of Medicine, for assistance in flow cytometry data acquisition and analysis.

Funding. This study was supported by National Institutes of Health (NIH) grants R01DK125746 (D.F.), P30DK116073 (L.Y.), R01DK110183 (M.L.G.), R01DK135688 (M.N.P.), and R01DK084171 (G.W.W.). The Aurora 4L spectral flow cytometer was funded through NIH grant S10OD026859. The Zeiss confocal microscope was funded through NIH shared instrumentation grant S10OD016374. Human pancreatic islets (RRID: SAMN34130383) were provided by the National Institute of Diabetes and Digestive and Kidney Diseases–funded Integrated Islet Distribution Program (RRID: SCR _014387) at City of Hope (NIH grant U24DK098085).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. D.K., Z.G., A.W.M., and D.F. were responsible for immunophenotyping and data interpretation. D.K., D.C.S., and G.W.W. performed mouse experiments. D.K. and M.L.G. performed immunohistology. Z.G. was responsible for antibody production and β-cell experiments. S.Y. was responsible for biostatistics and data deposit. L.Y. performed insulin autoantibody measurement. C.S. and M.N.P. performed human islet immunolabeling and imaging. D.F. was responsible for conceptualization, experimental design, and manuscript writing. All authors read and edited the manuscript. D.F. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

This article contains supplementary material online at https://doi.org/10.2337/figshare.25254211.

This article is featured in a podcast available at diabetesjournals.org/diabetes/pages/diabetesbio.

1.
Ziegler
AG
,
Rewers
M
,
Simell
O
, et al
.
Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in children
.
JAMA
2013
;
309
:
2473
2479
2.
Vives
M
,
Somoza
N
,
Soldevila
G
, et al
.
Reevaluation of autoantibodies to islet cell membrane in IDDM. Failure to detect islet cell surface antibodies using human islet cells as substrate
.
Diabetes
1992
;
41
:
1624
1631
3.
Huang
Q
,
Merriman
C
,
Zhang
H
,
Fu
D.
Coupling of insulin secretion and display of a granule-resident zinc transporter ZnT8 on the surface of pancreatic beta cells
.
J Biol Chem
2017
;
292
:
4034
4043
4.
Guo
Z
,
Kasinathan
D
,
Merriman
C
, et al
.
Cell-surface autoantibody targets zinc transporter-8 (ZnT8) for in vivo β-cell imaging and islet-specific therapies
.
Diabetes
2023
;
72
:
184
195
5.
Gu
Y
,
Merriman
C
,
Guo
Z
, et al
.
Novel autoantibodies to the β-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes
.
J Autoimmun
2021
;
122
:
102677
6.
Merriman
C
,
Huang
Q
,
Gu
W
,
Yu
L
,
Fu
D.
A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells
.
J Biol Chem
2018
;
293
:
579
587
7.
Wan
H
,
Merriman
C
,
Atkinson
MA
, et al
.
Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform
.
Proc Natl Acad Sci U S A
2017
;
114
:
10196
10201
8.
Avalos
AM
,
Ploegh
HL.
Early BCR events and antigen capture, processing, and loading on MHC class II on B cells
.
Front Immunol
2014
;
5
:
92
9.
Lu
M
,
Fu
D.
Structure of the zinc transporter YiiP
.
Science
2007
;
317
:
1746
1748
10.
Batista
FD
,
Iber
D
,
Neuberger
MS.
B cells acquire antigen from target cells after synapse formation
.
Nature
2001
;
411
:
489
494
11.
Mariño
E
,
Tan
B
,
Binge
L
,
Mackay
CR
,
Grey
ST.
B-cell cross-presentation of autologous antigen precipitates diabetes
.
Diabetes
2012
;
61
:
2893
2905
12.
Brodie
GM
,
Wallberg
M
,
Santamaria
P
,
Wong
FS
,
Green
EA.
B-cells promote intra-islet CD8+ cytotoxic T-cell survival to enhance type 1 diabetes
.
Diabetes
2008
;
57
:
909
917
13.
Aguilar-Diosdado
M
,
Parkinson
D
,
Corbett
JA
, et al
.
Potential autoantigens in IDDM. Expression of carboxypeptidase-H and insulin but not glutamate decarboxylase on the beta-cell surface
.
Diabetes
1994
;
43
:
418
425
14.
Sanderson
NS
,
Zimmermann
M
,
Eilinger
L
, et al
.
Cocapture of cognate and bystander antigens can activate autoreactive B cells
.
Proc Natl Acad Sci U S A
2017
;
114
:
734
739
15.
Atkinson
MA
,
Bluestone
JA
,
Eisenbarth
GS
, et al
.
How does type 1 diabetes develop? The notion of homicide or β-cell suicide revisited
.
Diabetes
2011
;
60
:
1370
1379
16.
Thul
PJ
,
Åkesson
L
,
Wiking
M
, et al
.
A subcellular map of the human proteome
.
Science
2017
;
356
:
eaal3321
17.
Segerstolpe
Å
,
Palasantza
A
,
Eliasson
P
, et al
.
Single-cell transcriptome profiling of human pancreatic islets in health and type 2 diabetes
.
Cell Metab
2016
;
24
:
593
607
18.
Ravassard
P
,
Hazhouz
Y
,
Pechberty
S
, et al
.
A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion
.
J Clin Invest
2011
;
121
:
3589
3597
19.
Merriman
C
,
Fu
D.
Down-regulation of the islet-specific zinc transporter-8 (ZnT8) protects human insulinoma cells against inflammatory stress
.
J Biol Chem
2019
;
294
:
16992
17006
20.
Merriman
C
,
Li
H
,
Li
H
,
Fu
D.
Highly specific monoclonal antibodies for allosteric inhibition and immunodetection of the human pancreatic zinc transporter ZnT8
.
J Biol Chem
2018
;
293
:
16206
16216
21.
Mathews
CE
,
Xue
S
,
Posgai
A
, et al
.
Acute versus progressive onset of diabetes in NOD mice: potential implications for therapeutic interventions in type 1 diabetes
.
Diabetes
2015
;
64
:
3885
3890
22.
Takiishi
T
,
Cook
DP
,
Korf
H
, et al
.
Reversal of diabetes in NOD mice by clinical-grade proinsulin and IL-10–secreting Lactococcus lactis in combination with low-dose anti-CD3 depends on the induction of Foxp3-positive T cells
.
Diabetes
2017
;
66
:
448
459
23.
Kulkarni
RN
,
Mizrachi
EB
,
Ocana
AG
,
Stewart
AF.
Human β-cell proliferation and intracellular signaling: driving in the dark without a road map
.
Diabetes
2012
;
61
:
2205
2213
24.
Ehlers
MR.
Who let the dogs out? The ever-present threat of autoreactive T cells
.
Sci Immunol
2018
;
3
:
eaar6602
25.
Wardemann
H
,
Yurasov
S
,
Schaefer
A
,
Young
JW
,
Meffre
E
,
Nussenzweig
MC.
Predominant autoantibody production by early human B cell precursors
.
Science
2003
;
301
:
1374
1377
26.
Wenzlau
JM
,
Juhl
K
,
Yu
L
, et al
.
The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes
.
Proc Natl Acad Sci U S A
2007
;
104
:
17040
17045
27.
Culina
S
,
Lalanne
AI
,
Afonso
G
, et al;
ImMaDiab Study Group
.
Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors
.
Sci Immunol
2018
;
3
:
eaao4013
28.
Reichardt
P
,
Dornbach
B
,
Rong
S
, et al
.
Naive B cells generate regulatory T cells in the presence of a mature immunologic synapse
.
Blood
2007
;
110
:
1519
1529
29.
Shevach
EM.
Mechanisms of Foxp3+ T regulatory cell-mediated suppression
.
Immunity
2009
;
30
:
636
645
30.
Herold
KC
,
Bundy
BN
,
Long
SA
, et al;
Type 1 Diabetes TrialNet Study Group
.
An anti-CD3 antibody, teplizumab, in relatives at risk for type 1 diabetes
.
N Engl J Med
2019
;
381
:
603
613
31.
Chatenoud
L
,
Thervet
E
,
Primo
J
,
Bach
JF.
Anti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice
.
Proc Natl Acad Sci U S A
1994
;
91
:
123
127
32.
Chimienti
F
,
Devergnas
S
,
Favier
A
,
Seve
M.
Identification and cloning of a beta-cell-specific zinc transporter, ZnT-8, localized into insulin secretory granules
.
Diabetes
2004
;
53
:
2330
2337
33.
Lemaire
K
,
Ravier
MA
,
Schraenen
A
, et al
.
Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice
.
Proc Natl Acad Sci U S A
2009
;
106
:
14872
14877
34.
Kim
YK
,
Walters
JA
,
Moss
ND
, et al
.
Zinc transporter 8 haploinsufficiency protects against beta cell dysfunction in type 1 diabetes by increasing mitochondrial respiration
.
Mol Metab
2022
;
66
:
101632
35.
Flannick
J
,
Thorleifsson
G
,
Beer
NL
, et al;
Go-T2D Consortium
;
T2D-GENES Consortium
.
Loss-of-function mutations in SLC30A8 protect against type 2 diabetes
.
Nat Genet
2014
;
46
:
357
363
36.
Dwivedi
OP
,
Lehtovirta
M
,
Hastoy
B
, et al
.
Loss of ZnT8 function protects against diabetes by enhanced insulin secretion
.
Nat Genet
2019
;
51
:
1596
1606
37.
Friend
SH
,
Schadt
EE.
Translational genomics. Clues from the resilient
.
Science
2014
;
344
:
970
972
38.
Atkinson
MA.
Evaluating preclinical efficacy
.
Sci Transl Med
2011
;
3
:
96cm22
39.
Énée
É
,
Kratzer
R
,
Arnoux
JB
, et al
.
ZnT8 is a major CD8+ T cell-recognized autoantigen in pediatric type 1 diabetes
.
Diabetes
2012
;
61
:
1779
1784
40.
Koczwara
K
,
Bonifacio
E
,
Ziegler
AG.
Transmission of maternal islet antibodies and risk of autoimmune diabetes in offspring of mothers with type 1 diabetes
.
Diabetes
2004
;
53
:
1
4
41.
Warram
JH
,
Krolewski
AS
,
Gottlieb
MS
,
Kahn
CR.
Differences in risk of insulin-dependent diabetes in offspring of diabetic mothers and diabetic fathers
.
N Engl J Med
1984
;
311
:
149
152
42.
Harjutsalo
V
,
Reunanen
A
,
Tuomilehto
J.
Differential transmission of type 1 diabetes from diabetic fathers and mothers to their offspring
.
Diabetes
2006
;
55
:
1517
1524
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/journals/pages/license.