Introduction
Type 1 diabetes is a T cell mediated autoimmune disease characterised by exogenous insulin dependency resulting from the destruction of the insulin-producing beta cells. Pathogenesis of type 1 diabetes involves the breakdown of tolerance, autoantibody production and the activation of islet antigen-specific autoreactive CD4
+ T cells that in turn provide help for islet antigen-specific CD8
+ cytotoxic T cell responses [
1‐
3]. Genetic association studies in type 1 diabetes have identified risk variants in the key genes responsible for autoimmune responses against insulin-derived peptides (HLA class II and class I genes, and the insulin gene), as well as in genes with prominent roles in CD4
+ T cell function. Several studies have investigated proinflammatory Th1/Th17 responses in type 1 diabetes patients, reporting a Th17 bias among type 1 diabetes patients as compared with healthy individuals [
4‐
8]. CD4
+ T cells purified from the blood of newly diagnosed type 1 diabetes patients were skewed towards IL-17 secretion when compared with healthy controls [
5,
6]; and monocytes from type 1 diabetes patients were shown to spontaneously secrete the proinflammatory cytokines IL-6 and IL-1β that induce Th17 cells [
4]. There is evidence from mouse models for a role of the IL-21 pathway in type 1 diabetes [
9], but aside from the established
IL2-IL21 region association with type 1 diabetes risk [
10], we have only identified one published, recent study that reported increased frequencies of circulating CD4
+ T follicular helper (Tfh) cells together with enhanced expression of IL-21 in type 1 diabetes patients [
8]. This contrasts with several reports of increases in Tfh frequencies in other autoimmune diseases such as Sjogren’s syndrome, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myasthenia gravis, autoimmune thyroid disease, juvenile dermatomyositis and multiple sclerosis [
11].
In the present study, we have characterised the production of the key proinflammatory cytokines IL-21, IL-17 and IFN-γ in the peripheral T cell compartment of 69 type 1 diabetes patients and 61 healthy donors. We report here an increased production of IL-21 and, to a lesser extent, IL-17, but not IFN-γ, in memory CD4+ T effector (Teff) cells from type 1 diabetes patients as compared with healthy controls. Consistent with the increased production of IL-21, we also report an increased frequency of peripheral Tfh from an independent cohort of 30 long-standing type 1 diabetes patients and 32 healthy controls. Collectively, these findings suggest that Tfh cells and IL-21-mediated inflammation are involved in the pathogenesis of type 1 diabetes.
Discussion
Systemic alterations in the peripheral immune system of autoimmune disease patients have proved challenging to identify, particularly in organ-specific autoimmune diseases such as type 1 diabetes, in which onset of the first disease symptoms, namely autoantibodies, can precede clinical diagnosis by many years. In the present study, following in vitro stimulation, we detected an increased proportion of IL-21-producing cells within the memory CD4
+ Teff population in type 1 diabetes patients. IL-21 is a well-established Tfh cytokine that plays a major role in the generation of germinal centre reactions and antibody production [
16], and Tfh cell differentiation has been previously shown to be negatively regulated by IL-2 [
17‐
19]. As a consequence, the demonstration of increased IL-21 production in type 1 diabetes patients supports the findings from genetic studies of type 1 diabetes implicating a dysfunction of the IL-2 signalling pathway in the pathogenesis of the disease [
20‐
22]. It will be important to determine if the variants mapping to the
IL2-IL21 region that influence susceptibility to type 1 diabetes and other autoimmune diseases [
10,
23‐
25] directly influence IL-2 or IL-21 production in a cell-intrinsic manner. In addition to promoting B cell responses, IL-21 has also been shown to be an important factor for the differentiation of the Th17 lineage [
6,
26,
27]; therefore, the increase in IL-17-producing cells in type 1 diabetes patients as compared with healthy controls observed in the current study, as well as in previous studies [
4‐
6], could be a consequence of increased IL-21 production. Results from a recent study emphasised the pleiotropic nature of IL-21: IL-21 promoted Th17 lineage differentiation and IL-10 production while inhibiting Th1 differentiation as well as the generation of potentially pathogenic Th1/17 effector cells [
14]. How these influences on T cell differentiation and effector function by IL-21 interact in an in vivo setting and contribute to autoimmune disease pathogenesis, especially balanced against the ability of IL-21 to promote B cell differentiation and antibody production, requires further investigation.
A limitation of the current study is that we are not able to provide a functional mechanism to support the suggested involvement of IL-21 in the aetiology of the disease. This question will need to be addressed in future mechanistic studies designed at characterising circulating Tfh cells in human type 1 diabetes patients in vivo, and exploring the clinical outcomes associated with the subset of type 1 diabetes patients with increased Tfh cell frequency and increased IL-21 levels. An intriguing possibility is that the effect of inherited genetic risk variants leading to deficient regulation of IL-2 signalling are manifested in the increased production of IL-21 by Tfh cells. Previously, we showed that variants in
IL2RA that predispose to type 1 diabetes reduce the level of IL-2RA/CD25 on Tregs and memory Teff cells [
20,
22], thereby potentially increasing the amount of homeostatic IL-2 production required for Treg survival and function, and limiting Tfh differentiation.
Overall, our findings underscore an inherent bias towards a proinflammatory response in type 1 diabetes patients and potentially reflect alterations of the immune system observed in the autoimmune microenvironment, which are critically dependent on IL-21 signalling [
28]. In agreement with this hypothesis, we show that the frequency of Tfh cells is increased on average by 14.9% in type 1 diabetes patients compared with controls, consistent with a recent study [
8]. The relevance of this Tfh phenotype to type 1 diabetes pathogenesis is strongly supported by previous reports showing increased levels of Tfh cells in seven other autoimmune diseases [
11]. Their increased prevalence in the circulation in other diseases also reduces the possibility that this Tfh phenotype, as well as the increased IL-21 production in type 1 diabetes patients shown here and previously reported [
8], are a consequence of insulin treatment, a potential confounding factor in type 1 diabetes case–control studies. One of the strongest type 1 diabetes associated loci,
PTPN22, may also be implicated in Tfh cell responses and IL-21 production leading to an increase in B cell numbers and antibody production in
Ptpn22 knockout mice [
29]. It is plausible that the increased frequency of circulating Tfh cells is directly responsible for the increased IL-21 production observed in type 1 diabetes patients. Of the 62 donors recalled for Tfh immunophenotyping, we were able to measure the frequency of IL-21
+ CD45RA
− T cells in 46 of them, and found a modest correlation between the frequencies of circulating Tfh and IL-21 CD45RA
− memory T cells. These data suggest that, although Tfh cells are likely an important source of this cytokine, there are also additional cell types that also contribute to IL-21 production, as previously described [
11].
In contrast to IL-21 and, to a lesser extent IL-17, we found no evidence for the differential expression of the proinflammatory cytokine IFN-γ in type 1 diabetes patients. This lack of association was observed not only among CD45RA
− CD4
+ Teff cells, but also among a subset of HELIOS
− CD45RA
− FOXP3
+ CD4
+ T cells. The ability of HELIOS
− FOXP3
+ CD4
+ T cells to produce IFN-γ is consistent with similar observations by McClymont et al who reported that HELIOS
− FOXP3
+ CD4
+ Tregs produced increased levels of IFN-γ in type 1 diabetes cases [
7].
In summary, we have identified an imbalance in IL-21 production in type 1 diabetes patients, which is accompanied by an increased frequency of circulating Tfh cells. Although this imbalance in the periphery is only manifested in a 21.9% increase in the frequency of IL-21
+ effector memory CD4
+ T cells in patients, it is likely the reflection of a much larger effect on secondary lymphoid organs, particularly at disease diagnosis. In mice, IL-21 inhibits T cell IL-2 production and impairs Treg homeostasis [
30]. Taken together with findings in other autoimmune diseases [
11], there is genetic and immunological rationale for the initiation of clinical trials to investigate the safety and efficacy of anti-IL-21 therapy in RA patients (
www.clinicaltrials.gov - NCT01565408 and NCT01647451) and in other diseases, including type 1 diabetes. Interestingly, B cell depletion with a course of anti-CD20 therapy (rituximab) decreased circulating IL-21 levels and the frequency of Tfh cells in peripheral blood of type 1 diabetes patients, and preserved beta cell function in ten out of 20 patients [
8]. The authors proposed that the observed effects of anti-CD20 reflect close interactions between Tfh and B cells in germinal centres. For example, in mice, IL-21 is a potent inducer of the co-stimulatory molecule CD86 on B cells thereby promoting antigen presentation [
31]. In light of previous evidence of some clinical efficacy of anti-CD20 therapy in type 1 diabetes patients [
32], it is possible that pre-selected patients stratified on the basis of increased levels of IL-21, Tfh cells or with multiple circulating autoantibodies and IFN-γ production [
33] could benefit to a greater extent from clinical interventions, such as B cell depletion, IL-2 replacement or anti-IL-21 therapy.
Acknowledgements
We thank staff of the National Institute for Health Research (NIHR) Cambridge BioResource recruitment team for assistance with volunteer recruitment and K. Beer, T. Cook, S. Hall and J. Rice for blood sample collection. We thank C. Guy for D-GAP sample recruitment. We thank M. Woodburn and T. Attwood for their contribution to sample management. This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. We thank members of the NIHR Cambridge BioResource SAB and management committee for their support and the NIHR Cambridge Biomedical Research Centre for funding. Access to NIHR Cambridge BioResource volunteers and their data and samples is governed by the NIHR Cambridge BioResource SAB. Documents describing access arrangements and contact details are available at
www.cambridgebioresource.org.uk/. We also thank H. Stevens, P. Clarke, G. Coleman, S. Dawson, S. Duley, M. Maisuria-Armer and T. Mistry for preparation of PBMC samples.