Type 1A, or autoimmune, diabetes is characterized by the presence of a series of autoantibodies targeting islet molecules, including insulin, GAD65, IA-2, and ZnT8 (15
). The initial assay for islet autoantibodies measured cytoplasmic islet cell autoantibodies by indirect immunofluorescence, using frozen sections of human pancreata (16
). IAAs are not detected with the islet cell antibody assay, whereas GADAs, IA2As, and ZnT8As can be detected. In addition to the autoantibodies, which were present before the diagnosis of diabetes, almost all patients treated with insulin (including patients with type 2 diabetes) developed high levels (relative to levels of prediabetic and new-onset patients) of insulin antibodies (17
). Islet autoantibodies have a number of distinguishing characteristics. IAAs often are (but not always) the first autoantibody to appear in children followed from birth (18
), and at onset of diabetes, their levels are inversely related to age of diabetes onset (19
). GADAs change the least with age of onset. ZnT8As are rapidly lost after diabetes onset (21
), and IA2As are very specific, identifying a particularly high diabetes risk of prospectively followed nondiabetic children (22
). The molecules insulin and ZnT8 are, to a large extent, limited to islet cells, whereas GAD65 and IA-2 are distributed in multiple neuroendocrine tissues. Given these unique properties for each of the islet autoantibodies, it is perhaps not surprising that rituximab had a differential effect upon the measured antibodies, with the most dramatic suppression of insulin autoantibodies and antibodies.
Rituximab treatment has been reported to blunt primary and secondary antibody responses (23
). Because patients who entered into this study were treated with insulin prior to determination of IAAs, it is not possible to isolate the effect of rituximab on pre-existing IAAs versus prevention and suppression of insulin antibodies induced by subcutaneous insulin therapy. This highlights the need for standardized assays that would distinguish the two types of antibodies. This may be particularly important given recent findings indicating that levels of IAAs, but not GADAs, and IA2As are inversely correlated with the rate at which prediabetic children progress to diabetes (25
A number of studies have documented the differential effects of anti-CD20 treatment on different antibodies in both humans and animal models. In general, rituximab therapy does not suppress overall IgG compared with its effect on IgM antibodies (12
). IgG levels can remain unaltered, whereas specific pathogenic autoantibodies are markedly suppressed. Specific disease-associated autoantibodies that are produced in sites outside of the bone marrow may be more susceptible to anti-CD20 suppression, and this may relate to the differential effect in the current study upon IAAs (5
). There is a large body of evidence in the NOD mouse model that insulin may be a primary autoantigen (26
). In particular, mutating an antigenic insulin peptide prevents diabetes in NOD mice (27
), whereas knockouts of both GAD65 and IA-2 do not influence progression to diabetes (28
). In addition, a polymorphism of the insulin gene correlated with increased levels of thymic insulin message is associated with decreased type 1A diabetes risk (30
Rituximab is remarkably effective in blocking de novo antibody responses and may be able to suppress both IAAs and insulin antibodies (the latter induced by injections of human insulin). In particular, 40% of patients treated with rituximab in the trial became negative (for variable times) for insulin antibodies/IAAs. Studies prior to, or within several weeks of, insulin therapy, given current insulin autoantibody/antibody assays, are needed to define whether rituximab can specifically suppress IAAs.
A significantly lower level of IAAs was observed, independent of rituximab treatment, in subjects defined as C-peptide responders versus nonresponders, particularly evident even in the placebo group. IA2As at entry also were found to be significantly lower (although not as dramatic a difference compared with IAAs) at entry in the placebo group for responders versus nonresponders, whereas no difference was observed for either GADAs or ZnT8As. The levels of IAAs were remarkably lower in responders compared with nonresponders at time 0 for both rituximab- and placebo-injection groups, which may indicate that the levels of IAAs, independent of rituximab treatment, are likely to be associated with disease prognosis or disease progression in patients with newly diagnosed type 1A diabetes. The relationship between the levels of IAAs and disease progression only has been investigated in the prediabetic period with the presence of anti-islet autoantibodies, but this has not been possible to study after disease onset. Among these biochemically defined islet autoantibodies, IAAs are the only ones in which the level is associated with the time to develop clinical type 1A diabetes among multiple autoantibody-positive prediabetic subjects (all followed to diabetes) in the Diabetes Autoimmunity Study in the Young (DAISY) study (33
). Higher IAA levels are associated with faster progression to overt diabetes. Combining this previous study with the present observation, IAA level seems to play a role in the prediction of disease progression, and higher levels of IAAs might indicate more aggressive autoimmune destruction of pancreatic β-cells both before and after clinical onset of diabetes. The IAAs in the current study are presumably a mixed population of naturally occurring IAAs and induced insulin antibodies. It needs to be further explored whether only IAAs or both IAAs and insulin antibodies secondary to injected insulin are associated with disease progression and the potentially therapeutic effect of rituximab.