Understanding and preventing type 1 diabetes through the unique working model of TrialNet

From a clinical standpoint, it is obvious that type 1 diabetes is a heterogeneous disease. For example, it can manifest very aggressively in a 3-year-old child who has rapid symptomatic onset, type-1-diabetes-associated autoantibodies, exceedingly low C-peptide levels, disease-predisposing HLA genes and an associated inflammatory process such as coeliac disease. But type 1 diabetes can also occur in a 40-year-old individual with limited HLA risk, detectable C-peptide, a mild, indolent onset and no other autoimmune diseases. With insulin replacement by injection being the only approved therapy for type 1 diabetes, there has seemed little need to identify disease heterogeneity from a clinical–pathological perspective [8]. However, this heterogeneity is emerging as a potentially important concept. Data from the T1D Exchange Clinic Network, for instance, demonstrated heterogeneity in loss of insulin secretion at each stage of disease [9]. Other studies, especially those of pancreas samples post-mortem in the Network for Pancreatic Donor (nPOD) collection, revealed considerable variability in the degree of residual insulin-reactive staining in recent-onset disease [10]. This prompted a more focused set of questions around whether endotypes exist within type 1 diabetes, which could have dramatic impact on our understanding of disease processes and therapeutic approaches. An endotype is a disease subtype defined by a distinct functional or pathobiological mechanism [11]. Results generated in a TrialNet ancillary study took the first steps towards the description of type 1 diabetes endotypes, using two complementary analyses of tissue and blood to identify disease-related subphenotypes in individuals with type 1 diabetes. The blood-based study (including samples from TN01) showed that approximately one-half of the individuals, analysed close to diagnosis, had autoreactive T cell responses characterised by the proinflammatory cytokine IFN-γ, whereas one-half were distinguished as having IL-10 responses (typical of immune regulation) along with significantly fewer autoantibodies. In the tissue-based study, analysis of pancreases obtained close to disease diagnosis (through the nPOD network) showed that approximately one-half of the individuals had an inflammatory islet infiltration distinguished by high numbers of CD20⁺ B lymphocytes, whereas one-half had sparse infiltration and significantly fewer B lymphocytes [12]. Thus, this work shows the existence of blood and tissue phenotypes (inflammatory CD20^High vs regulatory CD20^Low) that are likely to be related to each other and to variable rates of disease progression. Despite the fact that formal proof of such a link is still lacking–since blood and pancreas samples were collected from distinct individuals–this study provides the first example of a pathophysiological basis for disease heterogeneity that may have direct relevance to stratification for therapeutic trials [12].

Another important gap in type 1 diabetes research remains the lack of biomarkers that reflect an authentic signature of beta cell death and/or stress. Resolving this issue may also improve our ability to optimally time immunomodulatory treatments and other forms of interventions in type 1 diabetes. Several investigators have recently developed methods for assessing beta cell death in vivo by measuring the relative amount of beta cell-derived insulin encoding DNA (INS DNA) in the circulation. This analysis involves quantitative PCR amplification of INS DNA, with epigenetic marks that identify the DNA source as cells that actively transcribe insulin. Since the only significant source of non-methylated INS DNA in the body is the beta cell, the level of unmethylated INS DNA in the circulation reflects the active rate of beta cell death [13]. A recent TrialNet ancillary study determined levels of beta cell killing using this approach in individuals at risk of type 1 diabetes (from TN01). This study demonstrated that beta cell death can be found before the onset of type 1 diabetes. Importantly, the study gave insight into the tempo of the disease: while episodes of beta cell killing associated with a decline in insulin secretion were detected in the prediabetes period, there was a dramatic increase in killing in the peri-diagnosis period [14]. Although the ‘unmethylated INS DNA’ assay has yet to be fully validated [15], these data support the notion that a blood test that measures beta cell death in the setting of type-1-diabetes-associated autoimmunity can potentially be used in the future as the first surrogate marker of its kind, able to monitor beta cell ‘health-status’ during prevention and intervention studies.

Beta cells, like all professional secretory cells, naturally undergo high levels of endoplasmic reticulum stress as a result of their normal secretory physiology [16]. A hallmark of beta cell endoplasmic reticulum dysfunction is the accumulation and secretion of inadequately processed proinsulin molecules [17]. Therefore, using the proinsulin/C-peptide ratio (PI/C) as a non-invasive marker of beta cell dysfunction could provide a means to identify individuals at risk of developing type 1 diabetes prior to the onset of massive beta cell destruction. Sims et al recently reported that in archived samples from autoantibody-positive individuals enrolled in the Pathway to Prevention study (TN01), fasting PI/C was significantly increased ∼12 months prior to onset of type 1 diabetes [18]. Elevations were most pronounced in children aged ≤10 years, whose median PI/C was threefold higher than that of individuals who did not progress to type 1 diabetes. However, even among the entire group, increased PI/C was associated with increased odds of progression to type 1 diabetes after correction for age and BMI [18]. PI/C remains likely to be an informative and useful component of type 1 diabetes risk prediction algorithms, as well as an important biomarker of beta cell dysfunction in treatment trials. In addition, these results were confirmed by recent data demonstrating that in situ (in pancreas sections from diabetes autoantibody-positive donors), insulin area and beta cell mass can be maintained prior to disease onset and that production of proinsulin increases [19]. Using high-resolution confocal microscopy, this study revealed a high accumulation of vesicles containing proinsulin in beta cells from autoantibody-positive donors. This suggests either a defect in proinsulin conversion or an accumulation of immature vesicles due to an increase in insulin demand and/or to a dysfunction in vesicular trafficking. Taken together, these data suggest that prevention during stage 1 of the disease, when beta cell mass is still intact, could be a successful therapeutic strategy.

It is now clear that rates of progression to clinical symptoms vary significantly among individuals [5]. Thus, it would be desirable to identify additional biomarkers that could improve prediction beyond that afforded by autoantibody positivity. A recent study provided initial evidence that levels of selected circulating microRNAs are associated with increased risk of disease progression [20]. Augmented levels of these microRNAs conferred increased risk in young autoantibody-positive individuals enrolled in the Pathway to Prevention study (TN01). Moreover, the levels of several of the microRNAs associated with disease progression correlated with multiple OGTT indices of beta cell function, including both glucose and C-peptide area under the curve, peak C-peptide, and composite metabolic scores. Of note, several of these microRNAs have known influence on beta cell function and insulin secretion. Additional studies are needed to validate and expand these findings, which can be correlated with immunological and beta cell stress/death markers.

TrialNet performed a clinical trial of immunological co-stimulation blockade using CTLA-4–immunoglobulin (abatacept) in individuals newly diagnosed with type 1 diabetes (TN09). Two years of continuous treatment was associated with a slower decline of beta cell function and the beneficial effect was sustained for at least 1 year after therapy cessation [21]. To better understand the mechanism of this therapeutic effect, Orban and colleagues conducted an ancillary study to analyse immune cell subsets in treated and control individuals, using polychromatic flow cytometry [22]. The study revealed that continuous co-stimulation blockade is associated with an inversion of conventional naive/memory CD4⁺ T cell subset status, marked by a significant reduction in circulating CD4⁺CD45R0⁺CD62L⁺ central memory (CM) T cells and concomitant increase in CD4⁺CD45R0⁻CD62L⁺ naive T cells. These changes reverted after treatment cessation, supporting a direct drug effect. Importantly, the study also revealed that in placebo-treated individuals there was an increase in the number of circulating CM CD4⁺ T cells and a decrease in CD4-naive/CM T cell ratio, significantly associated with a subsequent rise in the rate of C-peptide decline. Abatacept treatment significantly altered this association. Mechanistically, this implies that the effect of co-stimulation blockade on naive/CM status of CD4⁺ T cells is linked to its therapeutic effect [22]. These data are the first to reveal an immunological biomarker of C-peptide level decline in individuals in whom type 1 diabetes has recently developed. This marker could assist in the prediction of progression of immunological damage to beta cells at various stages of the disease and provides a strong rationale for early therapeutic interference in co-stimulation and generation/maintenance of immune memory in type 1 diabetes. Currently, a TrialNet study of abatacept is being conducted in those at stage 2 of disease (i.e. with two autoantibodies and normal glucose tolerance) (www.​trialnet.​org/​our-research/​prevention-studies) (TN18, Fig. 1b).

Type 1 diabetes is typically described as an immune-mediated disease in which T cells drive the specific destruction of pancreatic insulin-producing beta cells. Recent data arising from TrialNet ancillary studies indicate that this T cell-centric view may need broadening. The role of B lymphocytes in type 1 diabetes is poorly understood despite autoantibodies being known robust predictive biomarkers. From results of studies making use of TrialNet samples, it is now becoming apparent that a breach in B lymphocyte tolerance is also a major contributor to type 1 diabetes. The trigger for these findings is the success of TN05, a clinical trial in individuals with new-onset type 1 diabetes, using monoclonal anti-CD20 antibody therapy (rituximab) to deplete CD20⁺ B cells. Those receiving active treatment showed reduced decline in C-peptide secretion in the year following introduction of therapy and better glycaemic control with reduced requirements for exogenous insulin [23]. Disappointingly, however, clinical efficacy was not sustained much beyond 1 year, prompting initiation of several TrialNet ancillary studies designed to better understand this phenomenon [24]. Meffre and co-workers, who previously reported defective central and peripheral B cell tolerance checkpoints resulting in the accumulation of self-reactive mature B cells in individuals with type 1 diabetes [25], investigated whether anti-B cell therapy modifies the frequency of autoreactive B cells. They found that B cell depletion did not change the frequencies of autoreactive and polyreactive B cells, which remained elevated in all individuals after rituximab treatment. There was a limited proliferative history of autoreactive B cells after treatment but these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. Thus, anti-B cell therapy may provide a temporary reduction in autoimmune processes through B cell depletion. However, repletion with autoreactive B cells after this therapy may explain the relapse that occurs in many autoimmune individuals. This study adds weight to the emerging concept that autoreactive B cells have a major pathogenic role in human type 1 diabetes. Moreover, these and other studies support the idea of trialling sequential therapy: first depleting B cells and then addressing resurging T cell activation.

In accordance with this view, Cambier and colleagues demonstrated that high-affinity insulin-binding B cells are present in healthy individuals exclusively in the anergic naive compartment, while these insulin-binding B cells lose their anergic phenotype in individuals who are at risk of diabetes (TN01 participants) or who have new-onset (< 1 year) type 1 diabetes [26]. These findings suggest that disruption of B cell anergy may predispose individuals towards autoimmunity, consistent with a potential early role for B cells in the development of type 1 diabetes.

A further, often neglected, component of the immune responses in individuals with type 1 diabetes has been that of innate immune pathways. A careful examination of the peripheral blood of relatives of individuals with type 1 diabetes (TN01) and of individuals with established disease has revealed a reduced number of circulating neutrophils. Indeed, the higher the risk of progression to diabetes, the more pronounced the reduction. This observation was possible through direct access to fresh blood from donors participating in TN01 using the Living Biobank approach. The observed reduction in circulating neutrophils is accompanied by the novel finding that neutrophils infiltrate the pancreas of individuals with type 1 diabetes, studied as cadaveric organ donors, suggesting that a disturbance in peripheral neutrophil regulation arises in parallel with neutrophil pancreas marginalisation [27]. Reduced circulating neutrophils have also been found in individuals with type 1 diabetes (<100 days from diagnosis) enrolled in five different TrialNet intervention trials (TN02, TN05, TN08, TN09 and TN14; Fig. 1b) [28]. It remains to be defined whether this reduction may reflect the aetiopathology of disease or serve as a clinical biomarker of a subgroup of individuals who develop diabetes.

Additional evidence pointing to the presence of innate immune mechanisms during the early stage of the disease (stage 1) comes from Hessner’s group [29]. They used plasma-induced transcriptional changes to investigate immune states within type 1 diabetes families (TN01 donors) and how these alter over time with either progression or non-progression to diabetes. While the plasma of individuals with recent-onset diabetes induced a distinct signature relative to that of control non-diabetic individuals and those at low or high risk of developing type 1 diabetes (i.e. autoantibody-negative siblings who either lacked or possessed HLA-DR3 and/or -DR4 haplotypes, respectively), the signatures of these healthy cohorts were also distinct from one another [30]. Notably, the signature of low-risk individuals exhibited the most robust induction of innate inflammatory transcripts (e.g. IL-1B, chemokine [C-C motif] ligand 2 [CCL2], CCL3, CCL7, chemokine [C-X-C motif] ligand 1 [CXCL1], CXCL2, CXCL3, CD14 and triggering receptor expressed on myeloid cells 1 [TREM1]). Pathway analyses revealed a continuum of immune states across the four cohorts. The plasma of individuals with recent-onset type 1 diabetes induced signatures intermediate between those at low and high risk of developing the disease, such that inflammation decreased and regulation increased across the low risk → recent-onset disease → high risk → non-diabetic donor continuum [30]. These results not only shed some light on the importance of innate immunity pathways in the early stage of the disease but they may also present new therapeutic opportunities before autoantibody development.